Compounds

ABSTRACT

A compound of Formula (I) 
                         
wherein Y 1 , Y 2 , X 1 , X 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , and R 9  are as defined herein;
 
and pharmaceutically acceptable salts and tautomers thereof, compositions, combinations and medicaments containing said compounds and processes for their preparation. The invention also relates to the use of said compounds, combinations, compositions and medicaments, in the treatment of diseases in which modulation of STING (Stimulator of Interferon Genes) is beneficial, for example inflammation, allergic and autoimmune diseases, infectious diseases, cancer, pre-cancerous syndromes and as vaccine adjuvants.

This application is a continuation of U.S. application Ser. No.15/586,634 filed on May 4, 2017, which is a continuation of U.S.application Ser. No. 15/368,921 filed on Dec. 5, 2016, and issued onAug. 1, 2017, U.S. Pat. No. 9,718,848, which is a continuation ofPCT/IB2016/057265 filed on Dec. 1, 2016, which claims the benefit ofU.S. Provisional 62/332,517 filed on May 6, 2016, U.S. Provisional62/327,579 filed on Apr. 26, 2016, U.S. Provisional 62/299,704 filed onFeb. 25, 2016, U.S. Provisional 62/299,253 filed on Feb. 24, 2016, andU.S. Provisional 62/262,668 filed on Dec. 3, 2015, all of which areincorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, combinationsand medicaments containing said compounds and processes for theirpreparation. The invention also relates to the use of said compounds,combinations, compositions and medicaments, in the treatment of diseasesin which modulation of STING (Stimulator of Interferon Genes) isbeneficial, for example inflammation, allergic and autoimmune diseases,infectious diseases, human immunodeficiency virus (HIV) infection, AIDS,cancer, pre-cancerous syndromes and as immugenic composition or vaccineadjuvants.

BACKGROUND TO THE INVENTION

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defence to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and adaptive immunity. The innate immune system is the firstline of defence which is initiated by Pattern Recognition Receptors(PRRs) which detect ligands from the pathogens as well as damageassociated molecular patterns (Takeuchi O. et al, Cell, 2010: 140,805-820). A growing number of these receptors have been identifiedincluding Toll-like receptors (TLRs), C-type lectin receptors, retinoicacid inducible gene I (RIG-I)-like receptors and NOD-like receptors(NLRs) and also double stranded DNA sensors. Activation of PRRs leads toup-regulation of genes involved in the inflammatory response includingtype 1 interferons, pro-inflammatory cytokines and chemokines whichsuppress pathogen replication and facilitate adaptive immunity.

The adaptor protein STING (Stimulator of Interferon Genes), also knownas TMEM 173, MPYS, MITA and ERIS, has been identified as a centralsignalling molecule in the innate immune response to cytosolic nucleicacids (Ishikawa H and Barber G N, Nature, 2008: 455, 674-678;WO2013/1666000). Activation of STING results in up-regulation of IRF3and NFκB pathways leading to induction of Interferon-β and othercytokines. STING is critical for responses to cytosolic DNA of pathogenor host origin, and of unusual nucleic acids called Cyclic Dinucleotides(CDNs)

CDNs were first identified as bacterial secondary messengers responsiblefor controlling numerous responses in the prokaryotic cell. BacterialCDNs, such as c-di-GMP are symmetrical molecules characterised by two3′,5′ phophodiester linkages.

Direct activation of STING by bacterial CDNs has recently been confirmedthrough X-ray crystallography (Burdette D L and Vance R E, NatureImmunology, 2013: 14, 19-26). Bacterial CDNs and their analogues haveconsequently attracted interest as potential vaccine adjuvants (LibanovaR. et al, Microbial Biotechnology 2012: 5, 168-176; WO2007/054279,WO2005/087238).

More recently, the response to cytosolic DNA has been elucidated andshown to involve generation, by an enzyme called cyclic GMP-AMP synthase(cGAS, previously known as C6orf150 or MB21D1), of a novel mammalian CDNsignalling molecule identified as cGAMP, which then activates STING.Unlike bacterial CDNs, cGAMP is an unsymmetrical molecule characterisedby it's mixed 2′,5′ and 3′,5′ phosphodiester linkages. (Gao P et al,Cell, 2013: 153, 1-14). Interaction of cGAMP with STING has also beendemonstrated by X-ray crystallography (Cai X et al, Molecular Cell,2014: 54, 289-296).

Interferon was first described as a substance which could protect cellsfrom viral infection (Isaacs & Lindemann, J. Virus Interference. Proc.R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267). In man, the type Iinterferons are a family of related proteins encoded by genes onchromosome 9 and encoding at least 13 isoforms of interferon alpha(IFNα) and one isoform of interferon beta (IFNβ). Recombinant IFNα wasthe first approved biological therapeutic and has become an importanttherapy in viral infections and in cancer. As well as direct antiviralactivity on cells, interferons are known to be potent modulators of theimmune response, acting on cells of the immune system.

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines, could become an important strategy for thetreatment or prevention of human diseases including viral infections.This type of immunomodulatory strategy has the potential to identifycompounds which may be useful not only in infectious diseases but alsoin cancer (Zitvogel, L., et al., Nature Reviews Immunology, 2015 15(7),p 405-414), allergic diseases (Moisan J. et al, Am. J. Physiol. LungCell Mol. Physiol., 2006: 290, L987-995), other inflammatory conditionssuch as irritable bowel disease (Rakoff-Nahoum S., Cell., 2004, 23,118(2): 229-41), and as vaccine adjuvants (Persing et al. TrendsMicrobiol. 2002: 10(10 Suppl), S32-7 and Dubensky et al., TherapeuticAdvances in Vaccines, published on line Sep. 5, 2013).

Allergic diseases are associated with a Th2-biased immune-response toallergens. Th2 responses are associated with raised levels of IgE,which, via its effects on mast cells, promotes a hypersensitivity toallergens, resulting in the symptoms seen, for example, in allergicrhinitis and asthma. In healthy individuals the immune-response toallergens is more balanced with a mixed Th2/Th1 and regulatory T cellresponse. Induction of Type 1 interferons have been shown to result inreduction of Th2-type cytokines in the local environment and promoteTh1/Treg responses. In this context, induction of type 1 interferons by,for example, activation of STING, may offer benefit in treatment ofallergic diseases such as asthma and allergic rhinitis (Huber J. P. etal J Immunol 2010: 185, 813-817).

In contrast, increased and prolonged type I IFN production is associatedwith a variety of chronic infections, including Mycobacteria (Collins etal, CHM 2015; Wassermann et al., CHM 2015; Watson et al., CHM 2015),Franciscella (Storek et al., JI 2015; Jin et al., JI 2011), Chlamydia(Prantner et al., JI 2010; Barker et al., Mbio 2013; Zhang et al., JI2014), Plasmodium (Sharma et al., Immunity 2011) and HIV (Herzner etal., Nat Immunol 2015; Nissen et al., Clin Exp Immunol 2014; Gao et al.,Science 2013; Lahaye et al, Science 2013) (reviewed in Stiffer and Feng,JI 2014). Similarly, excess type I interferon production is found amongpatients with complex forms of autoimmune disease. Genetic evidence inhumans and support from studies in animal models support the hypothesisthat inhibition of STING results in reduced type I interferon thatdrives autoimmune disease (Crow Y J, et al., Nat. Genet. 2006;38917-920, Stetson D B, et al., Cell 2008; 134; 587-598). Therefore,inhibitors of STING provide a treatment to patients with chronic type Iinterferon and proinflammatory cytokine production associated withinfections or complex autoimmune diseases. Allergic diseases areassociated with a Th2-biased immune-response to allergens.

Compounds that bind to STING and act as agonist have been shown toinduce type 1 interferons and other cytokines on incubation with humanPBMCs. Compounds which induce human interferons may be useful in thetreatment of various diseases, for example the treatment of allergicdiseases and other inflammatory diseases for example allergic rhinitisand asthma, the treatment of infectious diseases, pre-canceroussyndromes and cancer, and may also be useful as immugenic composition orvaccine adjuvants. Compounds that bind to STING may act as antagonistsand could be useful in the treatment, for example of autoimmunediseases.

It is envisaged that targeting STING with activation or inhibitingagents may be a promising approach for treating diseases in whichmodulation for the type 1 IFN pathway is beneficial, includinginflammatory, allergic and autoimmune diseases, infectious diseases,cancer, pre-cancerous syndromes and as immugenic composition or vaccineadjuvants.

International Patent Applications WO2014/093936, WO2014/189805,WO2013/185052, U.S.2014/0341976, WO 2015/077354, PCT/EP2015/062281 andGB 1501462.4 disclose certain cyclic di-nucleotides and their use ininducing an immune response.

It is an object of the invention to provide further cyclicdi-nucleotides, suitably for the treatment of cancer.

SUMMARY OF THE INVENTION

The invention is directed to compounds according to Formula (I):

wherein Y¹, Y², X¹, X², R¹, R², R³, R⁴, R⁵, R⁶, R⁸, and R⁹ are asdefined below and pharmaceutically acceptable salts thereof.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof and one or more ofpharmaceutically acceptable excipients.

In a further aspect of the present invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereoffor use in therapy.

In a further aspect of the present invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereoffor use in the treatment of a disease in which modulation STING isbeneficial.

In a further aspect of the present invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereoffor use in the treatment of inflammation, allergic and autoimmunediseases, infectious diseases, cancer, pre-cancerous syndromes and asimmugenic composition or vaccine adjuvants.

In a further aspect of the present invention, there is provided a methodof the treatment of a disease in which modulation STING is beneficial ina subject comprising administering a therapeutically effective amount ofa compound of Formula (I) or a pharmaceutically acceptable salt thereof.

In a further aspect of the present invention, there is provided a methodof the treating inflammation, allergic and autoimmune diseases,infectious diseases and cancer in a subject comprising administering atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof.

In a further aspect of the present invention, there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof in the manufacture of a medicament for use the treatment of adisease in which modulation of STING is beneficial.

In a further aspect of the present invention, there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof in the manufacture of a medicament for use the treatment ofinflammation, allergic and autoimmune diseases, infectious diseases,pre-cancerous syndromes and cancer.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof and at least one furthertherapeutic agent and one or more of pharmaceutically acceptableexcipients.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent for use in therapy.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent for use in the treatment of adisease or condition in which modulation of STING is beneficial.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent for use in the treatment ofinflammation, allergic and autoimmune diseases, infectious diseases,pre-cancerous syndromes and cancer.

In a further aspect of the present invention, there is provided a methodof the treatment of a disease or condition in which modulation of STINGis beneficial in a subject comprising administering a therapeuticallyeffective amount of a a combination comprising a compound of Formula (I)or a pharmaceutically acceptable salt thereof and at least one furthertherapeutic agent.

In a further aspect of the present invention, there is provided a methodof the treatment of inflammation, allergic and autoimmune diseases,infectious diseases and cancer in a subject comprising administering atherapeutically effective amount of a combination comprising a compoundof Formula (I) or a pharmaceutically acceptable salt thereof and atleast one further therapeutic agent.

In a further aspect of the present invention, there is provided animmugenic composition or vaccine adjuvant comprising a compound ofFormula (I), or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention, there is provided a compositioncomprising a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, and one or more immunostimulatory agents.

In a further aspect of the present invention, there is provided animmunogenic composition comprising an antigen or antigen composition anda compound of Formula (I), or a pharmaceutically acceptable saltthereof.

In a further aspect of the present invention, there is provided animmunogenic composition comprising an antigen or antigen composition anda compound of Formula (I), or a pharmaceutically acceptable salt thereoffor use in the treatment or prevention of disease.

In a further aspect of the present invention, there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, for the manufacture of an immunogenic composition comprising anantigen or antigen composition, for the treatment or prevention ofdisease.

In a further aspect of the present invention, there is provided a methodof treating or preventing disease comprising the administration to ahuman subject suffering from or susceptible to disease, an immunogeniccomposition comprising an antigen or antigen composition and a compoundof Formula (I), or a pharmaceutically acceptable salt thereof.

In a further aspect of the present invention, there is provided animmugenic or vaccine composition comprising an antigen or antigencomposition and a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof for use in the treatment or prevention ofdisease.

In a further aspect of the present invention, there is provided animmugenic composition comprising an antigen or antigen composition and acompound of Formula (I), or a pharmaceutically acceptable salt thereoffor use in the treatment or prevention of disease.

In a further aspect of the present invention, there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, for the manufacture of an immugenic or vaccine compositioncomprising an antigen or antigen composition, for the treatment orprevention of disease.

In a further aspect of the present invention, there is provided a methodof treating or preventing disease comprising the administration to ahuman subject suffering from or susceptible to disease, a vaccinecomposition comprising an antigen or antigen composition and a compoundof Formula (I), or a pharmaceutically acceptable salt thereof.

In a further aspect there is provided a method of treating an HIVinfection, in a human having or at risk of having the infection byadministering to the human a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

In a further aspect there is provided a method of treating an AIDSinfection, in a human having the infection by administering to the humana therapeutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof.

In a further aspect there is provided a method of treating an HIVinfection in a human by administering to the human a therapeuticallyeffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structures of Compounds 1 to 13, where R⁷ is asdefined in Formula (I).

FIG. 2 depicts the structures of Compounds 14 to 26, where R⁷ is asdefined in Formula (I).

FIG. 3 depicts the structures of Compounds 27 to 39, where R⁷ is asdefined in Formula (I).

FIG. 4 depicts the structures of Compounds 40 to 42.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel compounds of Formula (I):

wherein:

Y¹ and Y² are independently CH₂ or O;

X¹ and X² are independently S or O;

R¹ is OH and R² is NH₂ or R¹ is NH₂ and R² is H;

R³ is OH and R⁴ is NH₂ or R³ is NH₂ and R⁴ is H;

R⁵ is selected from: F, OH, and OC(O)R⁷;

R⁶ is selected from: F, OH, and OC(O)R⁷;

-   -   provided: when neither R⁵ or R⁶ are F, at least one of Y¹ and Y²        is CH₂; and R⁸ and R⁹ are independently selected from: H,        CH₂OC(O)R⁷, CH₂OCO₂R⁷,    -   CH₂CH₂SC(O)R⁷, and CH₂CH₂SSCH₂R⁷;    -   provided: when both X¹ and X² are O, at least one of R⁸ and R⁹        is not H;        where R⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (I), at least one of Y¹ and Y² isO. Suitably in the compounds of Formula (I), both Y¹ and Y² are O.

Suitably in the compounds of Formula (I), at least one of X¹ and X² isS. Suitably in the compounds of Formula (I), X¹ is S. Suitably in thecompounds of Formula (I), both X¹ and X² are S.

Suitably in the compounds of Formula (I), R¹ is NH₂ and R² is H.

Suitably in the compounds of Formula (I), R⁵ is OH.

Suitably in the compounds of Formula (I), R⁶ is F.

Suitably in the compounds of Formula (I), when one of R⁸ and R⁹ is notH, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (I), when one of R⁸ and R⁹ is notH, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (I), when X¹ is S, R⁸ and R⁹ are H.

Suitably in the compounds of Formula (I), when X² is S, R⁸ and R⁹ are H.

Suitably in the compounds of Formula (I), one of R⁸ and R⁹ is H.

Suitably in the compounds of Formula (I), when X¹ and X² are 0, one ofR⁸ and R⁹ is H.

Suitably in the compounds of Formula (I), R⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (I), R⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (I), R⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (I), R⁷ is tert-butyl.

Suitably in the compounds of Formula (I), R⁷ is iso-propyl.

Examples of compounds of the present invention include the compoundsdepicted in FIGS. 1, 2, 3 and 4.

The compounds of Formula (I) may be in the form of a salt.

Included in the compounds of Formula (I) are compounds of Formula (II):

wherein:

Y¹¹ and Y¹² are independently CH₂ or O;

X¹¹ is S;

X¹² is O;

R¹¹ is OH and R¹² is NH₂ or R¹¹ is NH₂ and R¹² is H;

R¹³ is OH and R¹⁴ is NH₂ or R¹³ is NH₂ and R¹⁴ is H;

R¹⁵ is selected from: F, OH, and OC(O)R¹⁷;

R¹⁶ is selected from: F, OH, and OC(O)R¹⁷;

-   -   provided: when neither R¹⁵ or R¹⁶ are F, at least one of Y¹¹ and        Y¹² is CH₂; and

R¹⁸ and R¹⁹ are independently selected from: H, CH₂OC(O)R¹⁷, CH₂OCO₂R⁷,

-   -   CH₂CH₂SC(O)R¹⁷, and CH₂CH₂SSCH₂R¹⁷;        where R¹⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (II), at least one of Y¹¹ and Y¹²is O. Suitably in the compounds of Formula (I), both Y¹¹ and Y¹² are O.

Suitably in the compounds of Formula (II), R¹¹ is NH₂ and R¹² is H.

Suitably in the compounds of Formula (II), R¹⁵ is OH.

Suitably in the compounds of Formula (II), R¹⁶ is F.

Suitably in the compounds of Formula (II), when one of R¹⁸ and R¹⁹ isnot H, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (II), when one of R¹⁸ and R¹⁹ isnot H, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (II), R¹⁸ and R¹⁹ are H.

Suitably in the compounds of Formula (II), one of R¹⁸ and R¹⁹ is H.

Suitably in the compounds of Formula (II), R¹⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (II), R¹⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (II), R¹⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (II), R¹⁷ is tert-butyl.

Suitably in the compounds of Formula (II), R¹⁷ is iso-propyl.

Suitably the compounds of Formula (II) are in the form of apharmaceutically acceptable salt.

Included in the compounds of Formula (I) are compounds of Formula (III):

wherein:

Y²¹ and Y²² are independently CH₂ or O;

X²¹ is O;

X²² is S;

R²¹ is OH and R²² is NH₂ or R²¹ is NH₂ and R²² is H;

R²³ is OH and R²⁴ is NH₂ or R²³ is NH₂ and R²⁴ is H;

R²⁵ is selected from: F, OH, and OC(O)R²⁷;

R²⁶ is selected from: F, OH, and OC(O)R²⁷;

-   -   provided: when neither R²⁵ or R²⁶ are F, at least one of Y²¹ and        Y²² is CH₂; and

R²⁸ and R²⁹ are independently selected from: H, CH₂OC(O)R²⁷, CH₂OCO₂R²⁷,

-   -   CH₂CH₂SC(O)R²⁷, and CH₂CH₂SSCH₂R²⁷;        where R²⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (III), at least one of Y²¹ and Y²²is O. Suitably in the compounds of Formula (III), both Y²¹ and Y²² areO.

Suitably in the compounds of Formula (III), R²¹ is NH₂ and R²² is H.

Suitably in the compounds of Formula (III), R²⁵ is OH.

Suitably in the compounds of Formula (III), R²⁶ is F.

Suitably in the compounds of Formula (III), when one of R²⁸ and R²⁹ isnot H, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (III), when one of R²⁸ and R²⁹ isnot H, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (III), R²⁸ and R²⁹ are H.

Suitably in the compounds of Formula (III), one of R²⁸ and R²⁹ is H.

Suitably in the compounds of Formula (III), R²⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (III), R²⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (III), R²⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (III), R²⁷ is tert-butyl.

Suitably in the compounds of Formula (III), R²⁷ is iso-propyl.

Suitably the compounds of Formula (III) are in the form of apharmaceutically acceptable salt.

Included in the compounds of Formula (I) and the compounds of Formula(II) are compounds of Formula (IV):

wherein:

X³¹ is S;

X³² is O;

R³¹ is OH and R³² is NH₂ or R³¹ is NH₂ and R³² is H;

R³³ is OH and R³⁴ is NH₂ or R³³ is NH₂ and R³⁴ is H;

R³⁵ is selected from: F, OH, and OC(O)R³⁷;

R³⁶ is selected from: F, OH, and OC(O)R³⁷;

-   -   provided: at least one of R³⁵ and R³⁶ is F; and        R³⁸ and R³⁹ are independently selected from: H, CH₂OC(O)R³⁷,        CH₂OCO₂R³⁷,    -   CH₂CH₂SC(O)R³⁷, and CH₂CH₂SSCH₂R³⁷;        where R³⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (IV), R³¹ is NH₂ and R³² is H.

Suitably in the compounds of Formula (IV), R³⁵ is OH.

Suitably in the compounds of Formula (IV), R³⁶ is F.

Suitably in the compounds of Formula (IV), when one of R³⁸ and R³⁹ isnot H, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (IV), when one of R³⁸ and R³⁹ isnot H, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (IV), R³⁸ and R³⁹ are H.

Suitably in the compounds of Formula (IV), one of R³⁸ and R³⁹ is H.

Suitably in the compounds of Formula (IV), R³⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (IV), R³⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (IV), R³⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (IV), R³⁷ is tert-butyl.

Suitably in the compounds of Formula (IV), R³⁷ is iso-propyl.

Suitably the compounds of Formula (IV) are in the form of apharmaceutically acceptable salt.

Included in the compounds of Formula (I) and the compounds of Formula(III) are compounds of Formula (V):

wherein:

X⁴¹ is O;

X⁴² is S;

R⁴¹ is OH and R⁴² is NH₂ or R⁴¹ is NH₂ and R⁴² is H;

R⁴³ is OH and R⁴⁴ is NH₂ or R⁴³ is NH₂ and R⁴⁴ is H;

R⁴⁵ is selected from: F, OH, and OC(O)R⁴⁷;

R⁴⁶ is selected from: F, OH, and OC(O)R⁴⁷;

-   -   provided: at least one of R⁴⁵ and R⁴⁶ is F; and

R⁴⁸ and R⁴⁹ are independently selected from: H, CH₂OC(O)R⁴⁷, CH₂OCO₂R⁴⁷,

-   -   CH₂CH₂SC(O)R⁴⁷, and CH₂CH₂SSCH₂R⁴⁷;        where R⁴⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (V), R⁴¹ is NH₂ and R⁴² is H.

Suitably in the compounds of Formula (V), R⁴⁵ is OH.

Suitably in the compounds of Formula (V), R⁴⁶ is F.

Suitably in the compounds of Formula (V), when one of R⁴⁸ and R⁴⁹ is notH, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (V), when one of R⁴⁸ and R⁴⁹ is notH, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (V), R⁴⁸ and R⁴⁹ are H.

Suitably in the compounds of Formula (V), one of R⁴⁸ and R⁴⁹ is H.

Suitably in the compounds of Formula (V), R⁴⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (V), R⁴⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (V), R⁴⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (V), R⁴⁷ is tert-butyl.

Suitably in the compounds of Formula (V), R⁴⁷ is iso-propyl.

Suitably the compounds of Formula (V) are in the form of apharmaceutically acceptable salt.

Included in the compounds of Formula (I) are compounds of Formula (VI):

wherein:

X⁵¹ is O;

X⁵² is O;

R⁵¹ is OH and R⁵² is NH₂ or R⁵¹ is NH₂ and R⁵² is H;

R⁵³ is OH and R⁵⁴ is NH₂ or R⁵³ is NH₂ and R⁵⁴ is H;

R⁵⁵ is selected from: F, OH, and OC(O)R⁴⁷;

R⁵⁶ is F;

R⁵⁸ and R⁵⁹ are independently selected from: H, CH₂OC(O)R⁵⁷, CH₂OCO₂R⁵⁷,

-   -   CH₂CH₂SC(O)R⁵⁷, and CH₂CH₂SSCH₂R⁵⁷;        where R⁵⁷ is selected from: aryl, heteroaryl, heterocycloalkyl,        cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5        substituents independently selected from: aryl, cycloalkyl,        hydroxy and F;    -   provided that at least one of R⁵⁸ and R⁵⁹ is not H.        and pharmaceutically acceptable salts thereof.

Suitably in the compounds of Formula (VI), R⁵¹ is NH₂ and R⁵² is H.

Suitably in the compounds of Formula (VI), R⁵⁵ is OH.

Suitably in the compounds of Formula (VI), when one of R⁵⁸ and R⁵⁹ isnot H, it is CH₂CH₂SC(O)C₁₋₆alkyl.

Suitably in the compounds of Formula (VI), when one of R⁵⁸ and R⁵⁹ isnot H, it is CH₂CH₂SSC₁₋₄alkylOH.

Suitably in the compounds of Formula (VI), one of R⁵⁸ and R⁵⁹ is H.

Suitably in the compounds of Formula (VI), R⁵⁷ is C₁₂₋₁₈alkyl.

Suitably in the compounds of Formula (VI), R⁵⁷ is selected from:C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted with one to 5 substituentsindependently selected from: aryl, cycloalkyl and F.

Suitably in the compounds of Formula (VI), R⁵⁷ is C₁₋₂₀alkyl.

Suitably in the compounds of Formula (VI), R⁵⁷ is tert-butyl.

Suitably in the compounds of Formula (VI), R⁵⁷ is iso-propyl.

Suitably the compounds of Formula (VI) are in the form of apharmaceutically acceptable salt.

-   -   Included in the compounds of Formula (I) are:

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 1;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 2;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 1;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 2;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 1;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 2;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione;

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 1; and

-   (1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,    isomer 2;    and pharmaceutically acceptable salts thereof.

It will be appreciated that Compound 2 is mixture of isomers asindicated below.

-   -   Isomers of Compound 2 are:

It will be appreciated that Compound 28 is mixture of isomers asindicated below.

-   -   Isomers of Compound 28 are:

It will be appreciated that compounds depicted, for example, by thestructure:

also exist in a protonated form, such as:

which represents the same compound.

It will be appreciated that compounds depicted, for example, by thestructure:

also exist in a protonated form, such as:

which represents the same compound.

It will be appreciated that compounds depicted, for example, by thestructure:

also exist in a protonated form, such as:

which represents the same compound.

Typically, the salts of the present invention are pharmaceuticallyacceptable salts. Salts encompassed within the term “pharmaceuticallyacceptable salts” refer to non-toxic salts of the compounds of thisinvention.

Salts, including pharmaceutically acceptable salts, are readily preparedby those of skill in the art.

Representative pharmaceutically acceptable acid addition salts include,but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate,bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate(camsylate), caprate (decanoate), caproate (hexanoate), caprylate(octanoate), cinnamate, citrate, cyclamate, digluconate,2,5-dihydroxybenzoate, disuccinate, dodecylsulfate (estolate), edetate(ethylenediaminetetraacetate), estolate (lauryl sulfate),ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate,fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate),glucoheptonate (gluceptate), gluconate, glucuronate, glutamate,glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate,hydrabamine (N,N′-di(dehydroabietyl)-ethylenediamine), hydrobromide,hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate,lactobionate, laurate, malate, maleate, malonate, mandelate,methanesulfonate (mesylate), methylsulfate, mucate,naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate(napsylate), nicotinate, nitrate, oleate, palmitate,p-aminobenzenesulfonate, p-aminosalicyclate, pamoate (embonate),pantothenate, pectinate, persulfate, phenylacetate,phenylethylbarbiturate, phosphate, polygalacturonate, propionate,p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate,sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate,tartrate, teoclate (8-chlorotheophyllinate), thiocyanate, triethiodide,undecanoate, undecylenate, and valerate.

Representative pharmaceutically acceptable base addition salts include,but are not limited to, aluminium,2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine),arginine, benethamine (N-benzylphenethylamine), benzathine(N,N′-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth,calcium, chloroprocaine, choline, clemizole (1-pchlorobenzyl-2-pyrrolildine-1′-ylmethylbenzimidazole), cyclohexylamine,dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine,dimethylethanolamine, dopamine, ethanolamine, ethylenediamine,L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium,meglumine (N-methylglucamine), piperazine, piperidine, potassium,procaine, quinine, quinoline, sodium, strontium, t-butylamine, and zinc.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the compounds of Formula (I).

The compounds of the invention may exist in solid or liquid form. Insolid form, compound of the invention may exist in a continuum of solidstates ranging from fully amorphous to fully crystalline. The term‘amorphous’ refers to a state in which the material lacks long rangeorder at the molecular level and, depending upon the temperature, mayexhibit the physical properties of a solid or a liquid. Typically suchmaterials do not give distinctive X-ray diffraction patterns and, whileexhibiting the properties of a solid, are more formally described as aliquid. Upon heating, a change from solid to liquid properties occurswhich is characterized by a change of state, typically second order(‘glass transition’). The term ‘crystalline’ refers to a solid phase inwhich the material has a regular ordered internal structure at themolecular level and gives a distinctive X-ray diffraction pattern withdefined peaks. Such materials when heated sufficiently will also exhibitthe properties of a liquid, but the change from solid to liquid ischaracterized by a phase change, typically first order (‘meltingpoint’).

The compounds of the invention may have the ability to crystallize inmore than one form, a characteristic, which is known as polymorphism(“polymorphs”). Polymorphism generally can occur as a response tochanges in temperature or pressure or both and can also result fromvariations in the crystallization process. Polymorphs can bedistinguished by various physical characteristics known in the art suchas x-ray diffraction patterns, solubility and melting point.

The compounds of Formula (I) may exist in solvated and unsolvated forms.As used herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute (in this invention, a compound ofFormula (I) or a salt) and a solvent. Such solvents, for the purpose ofthe invention, may not interfere with the biological activity of thesolute. The skilled artisan will appreciate that pharmaceuticallyacceptable solvates may be formed for crystalline compounds whereinsolvent molecules are incorporated into the crystalline lattice duringcrystallization. The incorporated solvent molecules may be watermolecules or non-aqueous such as ethanol, isopropanol, DMSO, aceticacid, ethanolamine, and ethyl acetate molecules. Crystalline latticeincorporated with water molecules are typically referred to as“hydrates”. Hydrates include stoichiometric hydrates as well ascompositions containing variable amounts of water.

It is also noted that the compounds of Formula (I) may form tautomers.‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. It is understoodthat all tautomers and mixtures of tautomers of the compounds of thepresent invention are included within the scope of the compounds of thepresent invention. For example and for absolute clarity, in thecompounds of Formula (I) when R¹ or R³ represent OH, the compounds willform the keto tautomer (═O).

While aspects for each variable have generally been listed aboveseparately for each variable this invention includes those compounds inwhich several or each aspect in Formula (I) is selected from each of theaspects listed above. Therefore, this invention is intended to includeall combinations of aspects for each variable.

Native CDN molecules can be sensitive to degradation byphosphodiesterases that are present in the blood, on the surface of hostcell or in the host cells, for example in antigen presenting cells, thattake up vaccine formulations that contain said native CDN molecules.Specific examples are the ectonucleotidiases, such as CD39, CD73 andENPP1 that reside on cell plasma membrane, facing into plasma, many ofwhich are known to degrade nucleotides, for example ATP is converted toAMP by both CD39 and ENPP1. Recently ENPP1 has been identified as amajor contributor to the degradation of CDNs possessing a 2′-5′phosphodiester linkage (Li, L., et al., 2014, Nature Chemical Biology,10(12), p 1043-1048). The potency of a CDN possessing STING agonistactivity would be diminished by such degradation, resulting in loweramount of induced expression of a signature molecule of innate immunity(e.g., IFN-beta) and hence afford weaker adjuvant potency. The presentinvention describes two different and complementary approaches that canbe employed to enhance and maintain the potency of described novel CDNs.As described in more detail in the following sections these are thesubstitution of sulphur for oxygen in the non-bridging positions of thephosphodiester and the use of a prodrug strategy to enhance cellpenetration and protect the CDN from degradation.

One aspect of the present invention relates to stereochemically defineddiasteroemers of cyclic purine mono- and dithio-diphosphatedinucleotides which induce STING-dependent TBK1 activation and theirmethods of preparation and use.

The present invention relates to methods for providing a potent STINGagonist capable of priming and maintaining a T cell response to tumorantigens either alone or in combination with other immuno-oncologyagents and to methods for providing adjuvant compositions. Thesecompositions are comprised of one or more cyclic purine dinucleotides ofFormula (I), wherein the cyclic purine dinucleotides present in thecomposition are substantially pure single mono-thiophosphatediasteromers or di-thiophosphate diastereomers, methods for themanufacture thereof, and methods for the use thereof to stimulate animmune response in an animal. The goal of both the single agent andvaccine formulation is to provide a combination of antigens andadjuvants capable of generating a sufficient population of memory Tcells and/or B cells to react quickly to a pathogen, tumor cell, etc.,bearing an antigen of interest.

Thiophosphates (also referred to as phosphorothioates) are a variant ofnormal nucleotides in which one of the nonbridging oxygens attached tophosphorus is replaced by a sulfur. A phosphorothioate linkage isinherently chiral. The skilled artisan will recognize that thethiophosphates in this structure may each exist in R or S form. Thus,Rp, and Sp, forms are possible at each phosphorus atom. In each case,preferred are substantially pure diastereomers of these molecules.Examples of such CDN thiophosphate molecules are depicted in FIGS. 1 to4 herein.

The term “prodrug” as used herein refers to a modification ofcontemplated compounds, wherein the modified compound is convertedwithin the body (e.g., in a target cell or target organ) back into theunmodified form through enzymatic or non-enzymatic reactions. In manycases the prodrug form is inactive or substantially less active than theparent non-prodrug form of the contemplated compounds. In certainembodiments, the hydroxyl on one ribose comprises a prodrug leavinggroup (Compounds 13, 26 and 39). In other embodiments the prodrug forminvolves derivatization of either one or both of the phosphates and/orthiophosphates (Compounds 3-12, 16-25 and 29-38). Prodrugs can modifythe physicochemical, biopharmaceutic, and pharmacokinetic properties ofdrugs. Reasons for prodrug development are typically poor aqueoussolubility, chemical instability, low oral bioavailability, lack ofblood brain barrier penetration, and high first pass metabolismassociated with the parent drug. Suitable prodrug moieties are describedin, for example, “Prodrugs and Targeted Delivery,” J. Rautico, Ed., JohnWiley & Sons, 2011. Prodrugs of phosphates which are of particularrelevance to the current invention are described by Wiemer, A. J., andWiemer, D. F. “Prodrugs of Phosphonates and Phosphates: Crossing theMembrane Barrier” in Topics of Current Chemistry, (2015) V 360, 115-160.

Preferred cyclic purine dinucleotides of the present invention includethe prodrugs di-phosphate CDNs (such as, Compounds 9, 10, 11 and 12 ofFIG. 1; Compounds 22, 23, 24 and 25 of FIG. 2; Compounds 35, 36, 37 and38 of FIG. 3 and Compounds 40, 41 and 42 of FIG. 4) the non-prodrugmonothiophospates (such as, Compounds 2, 15 and 28 of FIGS. 1-3) andnon-prodrug dithiophosphosphates (such as Compounds 1, 14 and 27 ofFIGS. 1-3) and prodrug forms of both the mono- (such as Compounds 7, 8,20, 21, 33 and 34 of FIGS. 1-3) and di-thiophosphate (such as Compounds3-6, 16-19, and 29-32 of FIGS. 1 to 3).

Definitions

As used herein, “a compound of the invention” includes all solvates,complexes, polymorphs, radiolabeled derivatives, tautomers,stereoisomers and optical isomers of the compounds of Formula (I) andsalts thereof.

As used herein, specific Compounds of the invention are designatednumerically according to the indication in the Figures. For example,Compound 2 is the compound in FIG. 1 with “2” under it, and Compound 27is the compound in FIG. 3 with “27” under it. Further “a” and “b”, etc.designations correspond to Isomer 1 and Isomer 2, etc., respectively.For example, “Compound 2a” is Isomer 1 of Compound 2, “Compound 27b” isIsomer 2 of Compound 27.

Unless otherwise defined, the designation “Isomer” or “Diastereomer” isan indication of the order in which a specified compound is eluted froma separation column under the specified conditions. The specifiedcompound with a shorter retention time on LCMS is designated “Isomer 1”or “Diastereomer 1”, the specified compound with a longer retention timeon LCMS is designated “Isomer 2” or “Diastereomer 2”, etc.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

The term “prophylaxis” includes prevention and refers to a measure orprocedure which is to prevent rather than cure or treat a disease.Preventing refers to a reduction in risk of acquiring or developing adisease causing at least one clinical symptom of the disease not todevelop in a subject that may be exposed to a disease causing agent or asubject predisposed to the disease in advance of disease outset.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and dosage forms which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, or other problem or complication, commensurate with areasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable excipients” includes alldiluents, carriers binders, glidants and other components ofpharmaceutical formulations with which the compound of the invention isadministered.

“Alkyl” refers to a hydrocarbon chain having the specified number of“member atoms”. For example, C₁-C₆ alkyl refers to an alkyl group havingfrom 1 to 6 member atoms. For example, C₁₂-C₁₈alkyl refers to an alkylgroup having from 12 to 18 member atoms. For example, C₁-C₂₀alkyl refersto an alkyl group having from 1 to 20 member atoms. Alkyl groups may besaturated, unsaturated, straight or branched. Representative branchedalkyl groups have one, two, or three branches. Examplary alkyl includesmethyl, ethyl, ethylene, propyl (n-propyl and isopropyl), butene, butyl(n-butyl, isobutyl, and t-butyl), pentyl and hexyl.

“Cycloalkyl”, unless otherwise defined, refers to a saturated orunsaturated non aromatic hydrocarbon ring system having from three toseven carbon atoms. Cycloalkyl groups are monocyclic or bicyclic ringsystems. For example, C₃-C₇ cycloalkyl refers to a cycloalkyl grouphaving from 3 to 7 member atoms. Examples of cycloalkyl as used hereininclude: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, cycloheptyl and spiro heptane.

“Aryl” refers to an aromatic hydrocarbon ring. Aryl groups aremonocyclic, bicyclic, and tricyclic ring systems having a total of fiveto fourteen ring member atoms, wherein at least one ring system isaromatic and wherein each ring in the system contains 3 to 7 memberatoms, such as phenyl, naphthalene, tetrahydronaphthalene and biphenyl.Suitably aryl is phenyl.

“Heteroaryl” refers to a monocyclic aromatic 4 to 8 member ringcontaining from 1 to 7 carbon atoms and containing from 1 to 4heteroatoms, provided that when the number of carbon atoms is 3, thearomatic ring contains at least two heteroatoms. Heteroaryl groupscontaining more than one heteroatom may contain different heteroatoms.Examplary heteroaryl includes: pyrrolyl, pyrazolyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furanyl, furazanyl,thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl,triazinyl, tetrazinyl.

“Heterocycloalkyl” refers to a saturated or unsaturated non-aromaticring containing 4 to 12 member atoms, of which 1 to 11 are carbon atomsand from 1 to 6 are heteroatoms. Heterocycloalkyl groups containing morethan one heteroatom may contain different heteroatoms. Heterocycloalkylgroups are monocyclic ring systems or a monocyclic ring fused with anaryl ring or to a heteroaryl ring having from 3 to 6 member atoms.Examplary heterocycloalkyl includes: pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, pyranyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothienyl, pyrazolidinyl, oxazolidinyl, oxetanyl, thiazolidinyl,piperidinyl, homopiperidinyl, piperazinyl, morpholinyl, thiamorpholinyl,1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-oxathiolanyl,1,3-oxathianyl, 1,3-dithianyl, 1,3oxazolidin-2-one, hexahydro-1H-azepin,4,5,6,7,tetrahydro-1H-benzimidazol, piperidinyl,1,2,3,6-tetrahydro-pyridinyl and azetidinyl.

“Heteroatom”, unless otherwise defined, refers to a nitrogen, sulphur oroxygen atom.

Compositions

While it is possible that, for use in therapy, the compound of theinvention may be administered as the raw chemical, it is possible topresent the compound of the invention as the active ingredient as apharmaceutical composition. Such compositions can be prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound. Accordingly, the invention further providespharmaceutical compositions comprising a compound of the invention andone or more pharmaceutically acceptable excipients. The excipient(s)must be acceptable in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof. In accordance with another aspect of the invention there isalso provided a process for the preparation of a pharmaceuticalcomposition including the agent, or pharmaceutically acceptable saltsthereof, with one or more pharmaceutically acceptable excipients. Thepharmaceutical composition can be for use in the treatment and/orprophylaxis of any of the conditions described herein.

Generally, the compound of the invention is administered in apharmaceutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like.

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient, vehicle or carrier. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions.

Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered once or more than once aday. Such pharmaceutical compositions may be prepared by any of themethods well known in the pharmacy art.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), vaginal or injectable (includingsubcutaneous, intramuscular, parenteral, intravenous or intradermal)route. Such compositions may be prepared by any method known in the artof pharmacy, for example by bringing into association the activeingredient with the carrier(s) or excipient(s).

In addition to the above described routes of administration for thetreatment of cancer, the pharmaceutical compositions may be adapted foradministration by intratumoral or peritumoral injection. Theintratumoral or peritumoral injection of a compound of the presentinvention directly into or adjacent to a single solid tumor is expectedto elicit an immune response that can attack and destroy cancer cellsthroughout the body, substantially reducing and in some casespermanently eliminating the tumor from the diseased subject. Theactivation of the immune system in this manner to kill tumors at aremote site is commonly known as the abscopal effect and has beendemonstrated in animals with multiple therapeutic modalities, (van derJeught, et al., Oncotarget, 2015, 6(3), 1359-1381). A further advantageof local or intratumoral or peritumoral administration is the ability toachieve equivalent efficacy at much lower doses, thus minimizing oreliminating adverse events that may be observed at much higher systemicdoses (Marabelle, A., et al., Clinical Cancer Research, 2014, 20(7), p1747-1756).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert excipient such as ethanol,glycerol, water and the like. Powders are prepared by reducing thecompound to a suitable fine size and mixing with a similarly preparedpharmaceutical excipient such as an edible carbohydrate, as, forexample, starch or mannitol. Flavouring, preservative, dispersing andcolouring agent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Excipients including glidants andlubricants such as colloidal silica, talc, magnesium stearate, calciumstearate or solid polyethylene glycol can be added to the powder mixturebefore the filling operation. A disintegrating or solubilizing agentsuch as agar-agar, calcium carbonate or sodium carbonate can also beadded to improve the availability of the medicament when the capsule isingested.

Moreover, when desired or necessary, excipients including suitablebinders, glidants, lubricants, sweetening agents, flavours,disintegrating agents and colouring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, suspensions, syrups and elixirs can beprepared in dosage unit form so that a given quantity contains apredetermined amount of the compound. Syrups can be prepared bydissolving the compound in a suitably flavoured aqueous solution, whileelixirs are prepared through the use of a non-toxic alcoholic vehicle.Suspensions can be formulated by dispersing the compound in a non-toxicvehicle. Solubilizers and emulsifiers such as ethoxylated isostearylalcohols and polyoxy ethylene sorbitol ethers, preservatives, flavoradditive such as peppermint oil or natural sweeteners or saccharin orother artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of the invention may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines. The compounds of the invention may also beadministered in the form of a nanoparticulate delivery vehicle of whichthere are multiple compositions and methods of preparation. Bothpolymeric nanoparticles and properly composed and sized liposomes areparticularly advantageous formulations for the treatment of cancer andin particular for the delivery of the compounds of the present inventionas they are preferentially targeted to the tumor and lymph nodes. Thesetargeting formulations have several potential advantages, these are:protecting the compounds of the present invention from degradation,increasing the amount of active agent at the site action and minimizingunwanted potential side effects as the result of excessive systemicexposure (Cai, Shuang et al., 2011 Advanced Drug Delivery Reviews, 2011,V 63, p 901-908). The potential utility of such approaches in theformulation of a CDN STING agonist has been demonstrated forformulations acting directly on tumors (Nakumura, T. et al., Journal ofControlled Release, 2015, V 216, p 149-157) and as use as an adjuvant(Hanson, M. et al., Journal of Clinical Investigation, 2015, V 125(6), p2532-2546). Furthermore there are multiple modes of administration(intratumoral, subcutaneous, intravenous, intraperitoneal andintramuscular) of nanoparticle and liposomal formulations which may beof special utility to the compounds of the present invention.Specifically, similar to native CDN molecules, those of the presentinvention may be sensitive to degradation by phosphodiesterases that arepresent in or on host cells, for example in antigen presenting cells.The potency of a compound of the present invention may be diminished bysuch degradation, resulting in lower amount of induced expression of asignature molecule of innate immunity (e.g., IFN-beta). Hence, thisdegradation may afford weaker potency as measured by the releaseIFN-beta release from PBMCs or of reduced vaccine potency, as defined bythe magnitude of a measured antigen-specific immune response.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspensions drops, gels or drypowders.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulphite, and taste-masking agents. Compositionsmay also be administered to the nose or other regions of the respiratorytract by nebulisation.

Intranasal compositions may permit the compound(s) of Formula (I) or (a)pharmaceutically acceptable salt(s) thereof to be delivered to all areasof the nasal cavities (the target tissue) and further, may permit thecompound(s) of Formula (I) or (a) pharmaceutically acceptable salt(s)thereof to remain in contact with the target tissue for longer periodsof time. A suitable dosing regime for intranasal compositions would befor the patient to inhale slowly through the nose subsequent to thenasal cavity being cleared. During inhalation the composition would beadministered to one nostril while the other is manually compressed. Thisprocedure would then be repeated for the other nostril. Typically, oneor two sprays per nostril would be administered by the above procedureone, two, or three times each day, ideally once daily. Of particularinterest are intranasal compositions suitable for once-dailyadministration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicel®(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an amount of from 0.001to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the total weightof the composition.

Compositions (for example wherein at least one compound is insuspension) may include one or more surfactants which functions tofacilitate dissolution of the medicament particles in the aqueous phaseof the composition. For example, the amount of surfactant used is anamount which will not cause foaming during mixing. Examples ofpharmaceutically acceptable surfactants include fatty alcohols, estersand ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity-adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity-adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

The compositions of the invention may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol.

In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. Such a fluid dispenser may have adispensing nozzle or dispensing orifice through which a metered dose ofthe fluid composition is dispensed upon the application of auser-applied force to a pump mechanism of the fluid dispenser. Suchfluid dispensers are generally provided with a reservoir of multiplemetered doses of the fluid composition, the doses being dispensable uponsequential pump actuations. The dispensing nozzle or orifice may beconfigured for insertion into the nostrils of the user for spraydispensing of the fluid composition into the nasal cavity. A fluiddispenser of the aforementioned type is described and illustrated inInternational Patent Application publication number WO 2005/044354(Glaxo Group Limited). The dispenser has a housing which houses afluid-discharge device having a compression pump mounted on a containerfor containing a fluid composition. The housing has at least onefinger-operable side lever which is movable inwardly with respect to thehousing to move the container upwardly in the housing by means of a camto cause the pump to compress and pump a metered dose of the compositionout of a pump stem through a nasal nozzle of the housing. In oneembodiment, the fluid dispenser is of the general type illustrated inFIGS. 30-40 of WO 2005/044354.

Aqueous compositions containing a compound of Formula (I) or apharmaceutically acceptable salt thereof may also be delivered by a pumpas disclosed in International Patent Application publication numberWO2007/138084 (Glaxo Group Limited), for example as disclosed withreference to FIGS. 22-46 thereof, or as disclosed in United Kingdompatent application number GB0723418.0 (Glaxo Group Limited), for exampleas disclosed with reference to FIGS. 7-32 thereof. The pump may beactuated by an actuator as disclosed in FIGS. 1-6 of GB0723418.0.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of the compound of Formula (I) or apharmaceutically acceptable salt thereof and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di-, orpolysaccharides (for example lactose or starch). Dry powder compositionsmay also include, in addition to the drug and carrier, a furtherexcipient (for example a ternary agent such as a sugar ester for examplecellobiose octaacetate, calcium stearate, or magnesium stearate.

In one embodiment, a composition suitable for inhaled administration maybe incorporated into a plurality of sealed dose containers provided onmedicament pack(s) mounted inside a suitable inhalation device. Thecontainers may be rupturable, peelable, or otherwise openableone-at-a-time and the doses of the dry powder composition administeredby inhalation on a mouthpiece of the inhalation device, as known in theart. The medicament pack may take a number of different forms, forinstance a disk-shape or an elongate strip. Representative inhalationdevices are the DISKHALER™ and DISKUS™ devices, marketed byGlaxoSmithKline.

A dry powder inhalable composition may also be provided as a bulkreservoir in an inhalation device, the device then being provided with ametering mechanism for metering a dose of the composition from thereservoir to an inhalation channel where the metered dose is able to beinhaled by a patient inhaling at a mouthpiece of the device. Exemplarymarketed devices of this type are TURBUHALER™ (AstraZeneca), TWISTHALER™(Schering) and CLICKHALER™ (Innovata.)

A further delivery method for a dry powder inhalable composition is formetered doses of the composition to be provided in capsules (one doseper capsule) which are then loaded into an inhalation device, typicallyby the patient on demand. The device has means to rupture, pierce orotherwise open the capsule so that the dose is able to be entrained intothe patient's lung when they inhale at the device mouthpiece. Asmarketed examples of such devices there may be mentioned ROTAHALER™(GlaxoSmithKline) and HANDIHALER™ (Boehringer Ingelheim.)

Pressurised aerosol compositions suitable for inhalation can be either asuspension or a solution and may contain a compound of Formula (I) or apharmaceutically acceptable salt thereof and a suitable propellant suchas a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixturesthereof, particularly hydrofluoroalkanes, especially1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or amixture thereof. The aerosol composition may optionally containadditional composition excipients well known in the art such assurfactants e.g. oleic acid, lecithin or an oligolactic acid orderivative thereof e.g. as described in WO 94/21229 and WO 98/34596(Minnesota Mining and Manufacturing Company) and co-solvents e.g.ethanol. Pressurised compositions will generally be retained in acanister (e.g. an aluminium canister) closed with a valve (e.g. ametering valve) and fitted into an actuator provided with a mouthpiece.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical compositions adapted for injectable administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents.

Antisense or RNA interference molecules may be administered to themammal in need thereof. Alternatively, constructs including the same maybe administered. Such molecules and constructs can be used to interferewith the expression of the protein of interest, e.g., histonedemethylase and as such, modify histone demethylation. Typicallydelivery is by means known in the art.

Antisense or RNA interference molecules can be delivered in vitro tocells or in vivo, e.g., to tumors of a mammal. Nodes of delivery can beused without limitations, including: intravenous, intramuscular,intraperitoncal, intra-arterial, local delivery during surgery,endoscopic, subcutaneous, and per os. Vectors can be selected fordesirable properties for any particular application. Vectors can beviral or plasmid. Adenoviral vectors are useful in this regard.Tissue-specific, cell-type specific, or otherwise regulatable promoterscan be used to control the transcription of the inhibitorypolynucleotide molecules. Non-viral carriers such as liposomes ornanospheres can also be used.

The compounds of Formula (I) and pharmaceutically acceptable saltsthereof may also be formulated to produce a composition for use as anadjuvant to modulate vacine activity. Such compositions may containantibody(ies) or antibody fragment(s) or an antigenic componentincluding but not limited to protein, DNA, live or dead bacteria and/orviruses or virus-like particles, together with one or more componentswith adjuvant activity including but not limited to aluminium salts, oiland water emulsions, heat shock proteins, lipid A preparations andderivatives, glycolipids, other TLR agonists such as CpG DNA or similaragents, cytokines such as GM-CSF or IL-12 or similar agents.

A therapeutically effective amount of the agent will depend upon anumber of factors including, for example, the age and weight of thesubject, the precise condition requiring treatment and its severity, thenature of the formulation, and the route of administration, and willultimately be at the discretion of the attendant physician orveterinarian. In particular, the subject to be treated is a mammal,particularly a human.

The agent may be administered in a daily dose. This amount may be givenin a single dose per day or more usually in a number (such as two,three, four, five or six) of sub-doses per day such that the total dailydose is the same.

Suitably, the amount of the compound of the invention administeredaccording to the present invention will be an amount selected from 0.01mg to 1 g per day (calculated as the free or unsalted compound).

The compounds of Formula (I) and pharmaceutically acceptable saltsthereof may be employed alone or in combination with other therapeuticagents. The compounds of Formula (I) and pharmaceutically acceptablesalts thereof and the other pharmaceutically active agent(s) may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order,by any convenient route in separate or combined pharmaceuticalcompositions.

The amounts of the compound(s) of Formula (I) or pharmaceuticallyacceptable salt(s) thereof and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. The compoundsof the present invention and further therapeutic agent(s) may beemployed in combination by administration simultaneously in a unitarypharmaceutical composition including both compounds. Alternatively, thecombination may be administered separately in separate pharmaceuticalcompositions, each including one of the compounds in a sequential mannerwherein, for example, the compound of the invention is administeredfirst and the other second and visa versa. Such sequentialadministration may be close in time (e.g. simultaneously) or remote intime. Furthermore, it does not matter if the compounds are administeredin the same dosage form, e.g. one compound may be administered topicallyand the other compound may be administered orally.

The combinations may be presented as a combination kit. By the term“combination kit” “or kit of parts” as used herein is meant thepharmaceutical composition or compositions that are used to administerthe combination according to the invention. When both compounds areadministered simultaneously, the combination kit can contain bothcompounds in a single pharmaceutical composition, such as a tablet, orin separate pharmaceutical compositions. When the compounds are notadministered simultaneously, the combination kit will contain eachcompound in separate pharmaceutical compositions either in a singlepackage or in separate pharmaceutical compositions in separate packages.

The combination kit can also be provided by instruction, such as dosageand administration instructions. Such dosage and administrationinstructions can be of the kind that are provided to a doctor, forexample by a drug product label, or they can be of the kind that areprovided by a doctor, such as instructions to a patient.

When the combination is administered separately in a sequential mannerwherein one is administered first and the other second or vice versa,such sequential administration may be close in time or remote in time.For example, administration of the other agent several minutes toseveral dozen minutes after the administration of the first agent, andadministration of the other agent several hours to several days afterthe administration of the first agent are included, wherein the lapse oftime is not limited. For example, one agent may be administered once aday, and the other agent may be administered 2 or 3 times a day, or oneagent may be administered once a week, and the other agent may beadministered once a day and the like.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredients(s) may be used in the form of salts,for example as alkali metal or amine salts or as acid addition salts, orprodrugs, or as esters, for example lower alkyl esters, or as solvates,for example hydrates, to optimise the activity and/or stability and/orphysical characteristics, such as solubility, of the therapeuticingredient. It will be clear also that, where appropriate, thetherapeutic ingredients may be used in optically pure form.

When combined in the same composition it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the composition and may be formulated foradministration. When formulated separately they may be provided in anyconvenient composition, conveniently, in such a manner as known for suchcompounds in the art.

When the compound of Formula (I) is used in combination with a secondtherapeutic agent active against the same disease, condition ordisorder, the dose of each compound may differ from that when thecompound is used alone. Appropriate doses will be readily appreciated bythose skilled in the art.

In one embodiment the mammal in the methods and uses of the presentinvention is a human.

The invention also provides a pharmaceutical composition comprising from0.5 to 1,000 mg of a compound of Formula (I) or pharmaceuticallyacceptable salt thereof and from 0.5 to 1,000 mg of a pharmaceuticallyacceptable excipient.

The compounds of the invention are useful in the treatment of diseasesin which modulation of STING is beneficial. This includes inflammation,allergic and autoimmune diseases, infectious diseases, cancer andpre-cancerous syndromes.

As modulators of the immune response the compounds of Formula (I) andpharmaceutically acceptable salts thereof may also be useful, asstand-alone or in combination as an adjuvant in the treatment ofdiseases in which modulation of STING is beneficial.

In one aspect, the disease or condition is inflammation, allergy andautoimmune disorders Autoimmune diseases associated include, but are notlimited to systemic lupus erythmatosus, Psoriasis, insulin-dependentdiabetes mellitus (IDDM), dermatomyositis, human immunodeficiency virus(HIV), AIDS, and Sjogren's syndrome (SS).

Inflammation represents a group of vascular, cellular and neurologicalresponses to trauma. Inflammation can be characterised as the movementof inflammatory cells such as monocytes, neutrophils and granulocytesinto the tissues. This is usually associated with reduced endothelialbarrier function and oedema into the tissues. Inflammation can beclassified as either acute or chronic. Acute inflammation is the initialresponse of the body to harmful stimuli and is achieved by the increasedmovement of plasma and leukocytes from the blood into the injuredtissues. A cascade of biochemical event propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolongedinflammation, known as chronic inflammation, leads to a progressiveshift in the type of cells which are present at the site of inflammationand is characterised by simultaneous destruction and healing of thetissue from the inflammatory process.

When occurring as part of an immune response to infection or as an acuteresponse to trauma, inflammation can be beneficial and is normallyself-limiting. However, inflammation can be detrimental under variousconditions. This includes the production of excessive inflammation inresponse to infectious agents, which can lead to significant organdamage and death (for example, in the setting of sepsis). Moreover,chronic inflammation is generally deleterious and is at the root ofnumerous chronic diseases, causing severe and irreversible damage totissues. In such settings, the immune response is often directed againstself-tissues (autoimmunity), although chronic responses to foreignentities can also lead to bystander damage to self tissues.

The aim of anti-inflammatory therapy is therefore to reduce thisinflammation, to inhibit autoimmunity when present and to allow for thephysiological process or healing and tissue repair to progress.

The agents may be used to treat inflammation of any tissue and organs ofthe body, including musculoskeletal inflammation, vascular inflammation,neural inflammation, digestive system inflammation, ocular inflammation,inflammation of the reproductive system, and other inflammation, asexemplified below.

Musculoskeletal inflammation refers to any inflammatory condition of themusculoskeletal system, particularly those conditions affecting skeletaljoints, including joints of the hand, wrist, elbow, shoulder, jaw,spine, neck, hip, knew, ankle, and foot, and conditions affectingtissues connecting muscles to bones such as tendons. Examples ofmusculoskeletal inflammation which may be treated with compounds of theinvention include arthritis (including, for example, osteoarthritis,rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acuteand chronic infectious arthritis, arthritis associated with gout andpseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis,tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis,myositis, and osteitis (including, for example, Paget's disease,osteitis pubis, and osteitis fibrosa cystic).

Ocular inflammation refers to inflammation of any structure of the eye,including the eye lids. Examples of ocular inflammation which may betreated with the compounds of the invention include blepharitis,blepharochalasis, conjunctivitis, dacryoadenitis, keratitis,keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, anduveitis.

Examples of inflammation of the nervous system which may be treated withthe compounds of the invention include encephalitis, Guillain-Barresyndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis,myelitis and schizophrenia.

Examples of inflammation of the vasculature or lymphatic system whichmay be treated with the compounds of the invention includearthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.

Examples of inflammatory conditions of the digestive system which may betreated with the compounds of the invention include cholangitis,cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis,inflammatory bowel disease (such as Crohn's disease and ulcerativecolitis), ileitis, and proctitis.

Examples of inflammatory conditions of the reproductive system which maybe treated with the compounds of the invention include cervicitis,chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis,orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis,vulvitis, and vulvodynia.

The agents may be used to treat autoimmune conditions having aninflammatory component. Such conditions include acute disseminatedalopecia universalise, Behcet's disease, Chagas' disease, chronicfatigue syndrome, dysautonomia, encephalomyelitis, ankylosingspondylitis, aplastic anemia, hidradenitis suppurativa, autoimmunehepatitis, autoimmune oophoritis, celiac disease, Crohn's disease,diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome,Grave's disease, Guillain-Barre syndrome, Hashimoto's disease,Henoch-Schönlein purpura, Kawasaki's disease, lupus erythematosus,microscopic colitis, microscopic polyarteritis, mixed connective tissuedisease, multiple sclerosis, myasthenia gravis, opsocionus myoclonussyndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritisnodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehaemolytic anemia, interstitial cystitis, lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

The agents may be used to treat T-cell mediated hypersensitivitydiseases having an inflammatory component. Such conditions includecontact hypersensitivity, contact dermatitis (including that due topoison ivy), uticaria, skin allergies, respiratory allergies (hayfever,allergic rhinitis) and gluten-sensitive enteropathy (Celliac disease).

Other inflammatory conditions which may be treated with the agentsinclude, for example, appendicitis, dermatitis, dermatomyositis,endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitissuppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis,otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis,pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi,transplant rejection (involving organs such as kidney, liver, heart,lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel,skin allografts, skin homografts, and heart valve xengrafts, serumsickness, and graft vs host disease), acute pancreatitis, chronicpancreatitis, acute respiratory distress syndrome, Sexary's syndrome,congenital adrenal hyperplasis, nonsuppurative thyroiditis,hypercalcemia associated with cancer, pemphigus, bullous dermatitisherpetiformis, severe erythema multiforme, exfoliative dermatitis,seborrheic dermatitis, seasonal or perennial allergic rhinitis,bronchial asthma, contact dermatitis, astopic dermatitis, drughypersensistivity reactions, allergic conjunctivitis, keratitis, herpeszoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, opticneuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonarytuberculosis chemotherapy, idiopathic thrombocytopenic purpura inadults, secondary thrombocytopenia in adults, acquired (autroimmine)haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia ofchildhood, regional enteritis, autoimmune vasculitis, multiplesclerosis, chronic obstructive pulmonary disease, solid organ transplantrejection, sepsis. Preferred treatments include treatment of transplantrejection, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemiclupus erythematosis, psoriasis, chronic pulmonary disease, andinflammation accompanying infectious conditions (e.g., sepsis).

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof for use in thetreatment of inflammation, allergy and autoimmune disease.

In a further aspect there is provided a method of treating inflammation,allergy and autoimmune disease comprising administering to a human inneed thereof a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt thereof.

In a further aspect there is provided the use of a compound of Formula(I) or a pharmaceutically acceptable salt thereof in the manufacture ofa medicament for the treatment of inflammation, allergy and autoimmunedisease.

In one aspect the disease to be treated is asthma.

The compounds of Formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of allergic disease,inflammatory disease, autoimmune disease, for example; antigenimmunotherapy, anti-histamines, steroids, NSAIDs, bronchodilators (e.g.beta 2 agonists, adrenergic agonists, anticholinergic agents,theophylline), methotrexate, leukotriene modulators and similar agents;monoclonal antibody therapy such as anti-lgE, anti-TNF, anti-IL-5,anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; receptor therapiese.g. entanercept and similar agents; antigen non-specificimmunotherapies (e.g. interferon or other cytokines/chemokines,cytokine/chemokine receptor modulators, cytokine agonists orantagonists, TLR agonists and similar agents).

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent useful in the treatment ofallergic disease, inflammation or autoimmune disease.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent useful in the treatment ofallergic disease, inflammation or autoimmune disease for use in therapy.

In a further aspect there is provided a combination comprising acompound of Formula (I) or pharmaceutically acceptable salt thereof andat least one one further therapeutic agent useful in the treatment ofallergic disease, inflammation or autoimmune disease, for use in thetreatment of allergic disease, inflammation or autoimmune disease.

In a further aspect there is provided the use of a combinationcomprising a compound of Formula (I) or a pharmaceutically acceptablesalt thereof and at least one one further therapeutic agent useful inthe treatment of allergic disease, inflammation or autoimmune diseaseinthe manufacture of a medicament for the treatment of allergic disease,inflammation or autoimmune disease.

In a further aspect there is provided a method of treating allergicdisease, inflammation or autoimmune disease comprising administering toa human in need thereof a therapeutically effective amount of acombination comprising a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof and at least one further therapeutic agentuseful in the treatment of allergic disease, inflammation or autoimmunedisease.

In a further aspect there is provided a pharmaceutical compositioncomprising a combination comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof and at least one furthertherapeutic agent useful in the treatment of allergic disease,inflammation or autoimmune disease and one or more of pharmaceuticallyacceptable excipients.

In one aspect the disease to be treated with such a combination isasthma.

In one aspect the disease or condition to be treated is cancer.

Examples of cancer diseases in which compounds of Formula (I), orpharmaceutically acceptable salts or solvates thereof may havepotentially beneficial antitumour effects include, but are not limitedto, cancers of the lung, bone, pancreas, skin, head, neck, uterus,ovaries, stomach, colon, breast, esophagus, small intestine, bowel,endocrine system, thyroid glad, parathyroid gland, adrenal gland,urethra, prostate, penis, testes, ureter, bladder, kidney or liver;rectal cancer; cancer of the anal region; carcinomas of the fallopiantubes, endometrium, cervix, vagina, vulva, renal pelvis, renal cell;sarcoma of soft tissue; myxoma; rhabdomyoma; fibroma; lipoma; teratoma;cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemagioma; hepatoma;fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia;lymphocytic lymphomas; primary CNS lymphoma; neoplasms of the CNS;spinal axis tumours; squamous cell carcinomas; synovial sarcoma;malignant pleural mesotheliomas; brain stem glioma; pituitary adenoma;bronchial adenoma; chondromatous hanlartoma; inesothelioma; Hodgkin'sDisease or a combination of one or more of the foregoing cancers.

Suitably the present invention relates to a method for treating orlessening the severity of cancers selected from the group consisting ofbrain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme,Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease,Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,medulloblastoma, head and neck, kidney, liver, melanoma, ovarian,pancreatic, adenocarcinoma, ductal adenocarcinoma, adenosquamouscarcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate,sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblasticT cell leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acutemyelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblasticT cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, mantlecell leukemia, multiple myeloma, megakaryoblastic leukemia, multiplemyeloma, acute megakaryocytic leukemia, promyelocytic leukemia,erythroleukemia, malignant lymphoma, hodgkins lymphoma, non-hodgkinslymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicularlymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulvalcancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma,esophageal cancer, salivary gland cancer, hepatocellular cancer, gastriccancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST(gastrointestinal stromal tumor) and testicular cancer.

Suitably the present invention relates to a method for treating orlessening the severity of pre-cancerous syndromes in a mammal, includinga human, wherein the pre-cancerous syndrome is selected from: cervicalintraepithelial neoplasia, monoclonal gammapathy of unknown significance(MGUS), myelodysplastic syndrome, aplastic anemia, cervical lesions,skin nevi (pre-melanoma), prostatic intraepithleial (intraductal)neoplasia (PIN), Ductal Carcinoma in situ (DCIS), colon polyps andsevere hepatitis or cirrhosis.

The compounds of the present invention may also be used as adjuvants toimprove the immune response raised to any given antigen and/or reducereactogenicity/toxicity in a patient, particularly a human, in needthereof. As such, a compound of this invention may be used incombination with vaccine compositions to modify, especially to enhance,the immune response for example by increasing the level or duration ofprotection and/or allowing a reduction in the antigenic dose.

The compounds of Formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more vaccines orimmunogenic antigens useful in the prevention or treatment of viralinfections. Such vaccines or immunogenic antigens include, withoutlimitation to pathogen derived proteins or particles such as attenuatedviruses, virus particles, and viral proteins typically used asimmunogenic substances. Examples of viruses and viral antigens include,without limitations to Polioviruses, Cioronaviridae and Coronaviruses,Rhinovirus (all subtypes), Adenoviruses (all subtypes), Hepatitis A,Hepatitis B, Hepatitis C, Hepatitis D, Human papillomavirus (includingall subtypes), Rabies viruses, Human T-cell lympotropic virus (allsubtypes), Rubella virus, Mumps virus, Coxsackie virus A (all subtypes),Cosackie virus B (all subtypes), human enteroviruses, herpesvirusesincluding cytomegalovirus, Epstein-Barr virus, human herepesviruses (allsubtypes), herpes simplex virus, varicella zoster virus, humanimmunodeficiency virus (HIV) (all subtypes), AIDS, Epstein-Barr virus,Reoviruses (all subtypes), Filoviruses including Marburg virus and Ebolavirus (all stains), Arenaviruses including Lymphocytic choriomeningitisvirus, Lassa virus, Junin virus, and Machupo virus, Arbovirusesincluding West Nile virus, Dengue viruses (all serotypes), Zika virus,Colorado tick fever virus, Sindbis virus, Togaviraidae, Flaviviridae,Bunyaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Poxvirusesincluding orthopoxvirus (variola virus, monkypox virus, vaccinia virus,cowpox virus), yatapoxviruses (tanapox virus, Yaba monkey tumor virus),parapoxvirus, molluscipoxvirus, Yellow fever, Hantaviruses includingHantaan, Seoul, Dobrava, Sin Nombre, Puumala, and Dobrava-like Saaremaa,human para influenza viruses and influenza viruses (all types), H1N1influenza and swine influenza viruses, respiratory syncytial virus (allsubgroups), rotaviruses including human rotaviruses A-E, bovinerotavirus, rhesus monkey rotavirus, Polyomaviruses including simianvirus 40, JC virus, BK virus, Coltiviruses, eyach virus, calciviruses,and Parvoviridae including dependovirus, parvovirus and erythrovirus.

The compounds of the present invention may also be useful in thetreatment of one or more diseases afflicting mammals which arecharacterized by cellular proliferation in the area of disordersassociated with neo-vascularization and/or vascular permeabilityincluding blood vessel proliferative disorders including arthritis(rheumatoid arthritis) and restenosis; fibrotic disorders includinghepatic cirrhosis and atherosclerosis; mesangial cell proliferativedisorders include glomerulonephritis, diabetic nephropathy, malignantnephrosclerosis, thrombotic microangiopathy syndromes, proliferativeretinopathies, organ transplant rejection and glomerulopathies; andmetabolic disorders include psoriasis, diabetes mellitus, chronic woundhealing, inflammation and neurodegenerative diseases.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof for use in thetreatment of a disease state selected from the group consisting of: HIV,HBV, HCV, influenza, skin warts, multiple sclerosis, allergicinflammation, and as an adjuvant.

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In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof for use in thetreatment of cancer and/or pre-cancerous syndromes.

In a further aspect there is provided a method of treating cancercomprising administering to a human in need thereof a therapeuticallyeffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof.

In a further aspect there is provided the use of a compound of Formula(I) or a pharmaceutically acceptable salt thereof in the manufacture ofa medicament for the treatment of cancer and/or pre-cancerous syndromes.

In one embodiment, the compound of the invention may be employed withother therapeutic methods of cancer treatment. In particular, inanti-neoplastic therapy, combination therapy with otherchemotherapeutic, hormonal, antibody agents as well as surgical and/orradiation treatments other than those mentioned above are envisaged.

In one embodiment, the further anti-cancer therapy is surgical and/orradiotherapy.

In one embodiment, the further anti-cancer therapy is at least oneadditional anti-neoplastic agent.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one anti-neoplastic agent.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one anti-neoplastic agent, for use in therapy.

In a further aspect there is provided a combination comprising acompound of Formula (I) or pharmaceutically acceptable salt thereof andat least one anti-neoplastic agent, for use in treating cancer and/orpre-cancerous syndromes.

In a further aspect there is provided the use of a combinationcomprising a compound of Formula (I) or a pharmaceutically acceptablesalt thereof and at least one anti-neoplastic agent, in the manufactureof a medicament for the treatment of cancer and/or pre-canceroussyndromes.

In a further aspect there is provided a method of treating cancer,comprising administering to a human in need thereof a therapeuticallyeffective amount of a combination comprising a compound of Formula (I)or a pharmaceutically acceptable salt thereof and at least oneanti-neoplastic agent.

In a further aspect there is provided a pharmaceutical compositioncomprising a combination comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof and at least one furthertherapeutic agent, particularly at least one anti-neoplastic agent andone or more of pharmaceutically acceptable carriers, diluents andexcipients.

Any anti-neoplastic agent that has activity versus a susceptible tumorbeing treated may be utilized in the combination. Typicalanti-neoplastic agents useful include, but are not limited to,anti-microtubule agents such as diterpenoids and vinca alkaloids;platinum coordination complexes; alkylating agents such as nitrogenmustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, andtriazenes; antibiotic agents such as anthracyclins, actinomycins andbleomycins; topoisomerase II inhibitors such as epipodophyllotoxins;antimetabolites such as purine and pyrimidine analogues and anti-folatecompounds; topoisomerase I inhibitors such as camptothecins; hormonesand hormonal analogues; signal transduction pathway inhibitors;non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents;proapoptotic agents; cell cycle signaling inhibitors; immuno-oncologyagents and immunostimulatory agents.

Anti-microtubule or Anti-mitotic Agents:

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of anti-microtubule agents include, but are notlimited to, diterpenoids and vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specificanti-cancer agents that operate at the G₂/M phases of the cell cycle. Itis believed that the diterpenoids stabilize the β-tubulin subunit of themicrotubules, by binding with this protein.

Disassembly of the protein appears then to be inhibited with mitosisbeing arrested and cell death following. Examples of diterpenoidsinclude, but are not limited to, paclitaxel and its analog docetaxel.

Paclitaxel, 5β,20-epoxy-1,2α,4,7β,10β,13α-hexa-hydroxytax-11-en-9-one4,10-diacetate 2-benzoate 13-ester with(2R,3S)—N-benzoyl-3-phenylisoserine; is a natural diterpene productisolated from the Pacific yew tree Taxus brevifolia and is commerciallyavailable as an injectable solution TAXOL®. It is a member of the taxanefamily of terpenes. Paclitaxel has been approved for clinical use in thetreatment of refractory ovarian cancer in the United States (Markman etal., Yale Journal of Biology and Medicine, 64:583, 1991; McGuire et al.,Ann. Intem, Med., 111:273, 1989) and for the treatment of breast cancer(Holmes et al., J. Nat. Cancer Inst., 83:1797,1991.) It is a potentialcandidate for treatment of neoplasms in the skin (Einzig et. al., Proc.Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastireet. al., Sem. Oncol., 20:56, 1990). The compound also shows potentialfor the treatment of polycystic kidney disease (Woo et. al., Nature,368:750. 1994), lung cancer and malaria. Treatment of patients withpaclitaxel results in bone marrow suppression (multiple cell lineages,Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide. 1998) related tothe duration of dosing above a threshold concentration (50 nM) (Kearns,C. M. et. al., Seminars in Oncology, 3(6) p. 16-23, 1995).

Docetaxel, (2R,3S)—N-carboxy-3-phenylisoserine,N-tert-butyl ester,13-ester with 5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate; is commercially available as aninjectable solution as TAXOTERE®. Docetaxel is indicated for thetreatment of breast cancer. Docetaxel is a semisynthetic derivative ofpaclitaxel q.v., prepared using a natural precursor,10-deacetyl-baccatin III, extracted from the needle of the European Yewtree.

Vinca alkaloids are phase specific anti-neoplastic agents derived fromthe periwinkle plant. Vinca alkaloids act at the M phase (mitosis) ofthe cell cycle by binding specifically to tubulin. Consequently, thebound tubulin molecule is unable to polymerize into microtubules.Mitosis is believed to be arrested in metaphase with cell deathfollowing. Examples of vinca alkaloids include, but are not limited to,vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleukoblastine sulfate, is commercially available asVELBAN® as an injectable solution. Although, it has possible indicationas a second line therapy of various solid tumors, it is primarilyindicated in the treatment of testicular cancer and various lymphomasincluding Hodgkin's Disease; and lymphocytic and histiocytic lymphomas.Myelosuppression is the dose limiting side effect of vinblastine.

Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commerciallyavailable as ONCOVIN® as an injectable solution. Vincristine isindicated for the treatment of acute leukemias and has also found use intreatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.Alopecia and neurologic effects are the most common side effect ofvincristine and to a lesser extent myelosupression and gastrointestinalmucositis effects occur.

Vinorelbine, 3′,4′-didehydro -4′-deoxy-C′-norvincaleukoblastine[R—(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)], commerciallyavailable as an injectable solution of vinorelbine tartrate(NAVELBINE®), is a semisynthetic vinca alkaloid. Vinorelbine isindicated as a single agent or in combination with otherchemotherapeutic agents, such as cisplatin, in the treatment of varioussolid tumors, particularly non-small cell lung, advanced breast, andhormone refractory prostate cancers. Myelosuppression is the most commondose limiting side effect of vinorelbine.

Platinum Coordination Complexes:

Platinum coordination complexes are non-phase specific anti-canceragents, which are interactive with DNA. The platinum complexes entertumor cells, undergo, aquation and form intra- and interstrandcrosslinks with DNA causing adverse biological effects to the tumor.Examples of platinum coordination complexes include, but are not limitedto, oxaliplatin, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available asPLATINOL® as an injectable solution. Cisplatin is primarily indicated inthe treatment of metastatic testicular and ovarian cancer and advancedbladder cancer.

Carboplatin, platinum, diammine[1,1-cyclobutane-dicarboxylate(2-)-O,O′], is commercially available asPARAPLATIN® as an injectable solution. Carboplatin is primarilyindicated in the first and second line treatment of advanced ovariancarcinoma.

Alkylating Agents:

Alkylating agents are non-phase anti-cancer specific agents and strongelectrophiles. Typically, alkylating agents form covalent linkages, byalkylation, to DNA through nucleophilic moieties of the DNA moleculesuch as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazolegroups. Such alkylation disrupts nucleic acid function leading to celldeath. Examples of alkylating agents include, but are not limited to,nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil;alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; andtriazenes such as dacarbazine.

Cyclophosphamide,2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidemonohydrate, is commercially available as an injectable solution ortablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent orin combination with other chemotherapeutic agents, in the treatment ofmalignant lymphomas, multiple myeloma, and leukemias.

Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commerciallyavailable as an injectable solution or tablets as ALKERAN®. Melphalan isindicated for the palliative treatment of multiple myeloma andnon-resectable epithelial carcinoma of the ovary. Bone marrowsuppression is the most common dose limiting side effect of melphalan.

Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, iscommercially available as LEUKERAN® tablets. Chlorambucil is indicatedfor the palliative treatment of chronic lymphatic leukemia, andmalignant lymphomas such as lymphosarcoma, giant follicular lymphoma,and Hodgkin's disease.

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially availableas MYLERAN® TABLETS. Busulfan is indicated for the palliative treatmentof chronic myelogenous leukemia.

Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is commerciallyavailable as single vials of lyophilized material as BiCNU®. Carmustineis indicated for the palliative treatment as a single agent or incombination with other agents for brain tumors, multiple myeloma,Hodgkin's disease, and non-Hodgkin's lymphomas.

Dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, iscommercially available as single vials of material as DTIC-Dome®.Dacarbazine is indicated for the treatment of metastatic malignantmelanoma and in combination with other agents for the second linetreatment of Hodgkin's Disease.

Antibiotic Anti-neoplastics:

Antibiotic anti-neoplastics are non-phase specific agents, which bind orintercalate with DNA. Typically, such action results in stable DNAcomplexes or strand breakage, which disrupts ordinary function of thenucleic acids leading to cell death. Examples of antibioticanti-neoplastic agents include, but are not limited to, actinomycinssuch as dactinomycin, anthrocyclins such as daunorubicin anddoxorubicin; and bleomycins.

Dactinomycin, also known as Actinomycin D, is commercially available ininjectable form as COSMEGEN®. Dactinomycin is indicated for thetreatment of Wilm's tumor and rhabdomyosarcoma.

Daunorubicin,(8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12naphthacenedione hydrochloride, is commercially available as a liposomalinjectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.Daunorubicin is indicated for remission induction in the treatment ofacute nonlymphocytic leukemia and advanced HIV associated Kaposi'ssarcoma.

Doxorubicin,(8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl,7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedionehydrochloride, is commercially available as an injectable form as RUBEX®or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatmentof acute lymphoblastic leukemia and acute myeloblastic leukemia, but isalso a useful component in the treatment of some solid tumors andlymphomas.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated froma strain of Streptomyces verticillus, is commercially available asBLENOXANE®. Bleomycin is indicated as a palliative treatment, as asingle agent or in combination with other agents, of squamous cellcarcinoma, lymphomas, and testicular carcinomas.

Topoisomerase II Inhibitors:

Topoisomerase II inhibitors include, but are not limited to,epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude, but are not limited to, etoposide and teniposide.

Etoposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-ethylidene-β-D-glucopyranoside], is commercially availableas an injectable solution or capsules as VePESID® and is commonly knownas VP-16. Etoposide is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of testicular andnon-small cell lung cancers.

Teniposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-thenylidene-β-D-glucopyranoside], is commercially availableas an injectable solution as VUMON® and is commonly known as VM-26.Teniposide is indicated as a single agent or in combination with otherchemotherapy agents in the treatment of acute leukemia in children.

Antimetabolite Neoplastic Agents:

Antimetabolite neoplastic agents are phase specific anti-neoplasticagents that act at S phase (DNA synthesis) of the cell cycle byinhibiting DNA synthesis or by inhibiting purine or pyrimidine basesynthesis and thereby limiting DNA synthesis. Consequently, S phase doesnot proceed and cell death follows. Examples of antimetaboliteanti-neoplastic agents include, but are not limited to, fluorouracil,methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4-(1H,3H) pyrimidinedione, is commerciallyavailable as fluorouracil. Administration of 5-fluorouracil leads toinhibition of thymidylate synthesis and is also incorporated into bothRNA and DNA. The result typically is cell death. 5-fluorouracil isindicated as a single agent or in combination with other chemotherapyagents in the treatment of carcinomas of the breast, colon, rectum,stomach and pancreas. Other fluoropyrimidine analogs include 5-fluorodeoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.

Cytarabine, 4-amino-1-β-D-arabinofuranosyl-2 (1H)-pyrimidinone, iscommercially available as CYTOSAR-U® and is commonly known as Ara-C. Itis believed that cytarabine exhibits cell phase specificity at S-phaseby inhibiting DNA chain elongation by terminal incorporation ofcytarabine into the growing DNA chain. Cytarabine is indicated as asingle agent or in combination with other chemotherapy agents in thetreatment of acute leukemia. Other cytidine analogs include5-azacytidine and 2′,2′-difluorodeoxycytidine (gemcitabine).

Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, iscommercially available as PURINETHOL®. Mercaptopurine exhibits cellphase specificity at S-phase by inhibiting DNA synthesis by an as of yetunspecified mechanism. Mercaptopurine is indicated as a single agent orin combination with other chemotherapy agents in the treatment of acuteleukemia. A useful mercaptopurine analog is azathioprine.

Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commerciallyavailable as TABLOID®. Thioguanine exhibits cell phase specificity atS-phase by inhibiting DNA synthesis by an as of yet unspecifiedmechanism. Thioguanine is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of acute leukemia. Otherpurine analogs include pentostatin, erythrohydroxynonyladenine,fludarabine phosphate, and cladribine.

Gemcitabine, 2′-deoxy-2′, 2′-difluorocytidine monohydrochloride(β-isomer), is commercially available as GEMZAR®. Gemcitabine exhibitscell phase specificity at S-phase and by blocking progression of cellsthrough the G1/S boundary. Gemcitabine is indicated in combination withcisplatin in the treatment of locally advanced non-small cell lungcancer and alone in the treatment of locally advanced pancreatic cancer.

Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino]benzoyl]-L-glutamic acid, is commercially available as methotrexatesodium. Methotrexate exhibits cell phase effects specifically at S-phaseby inhibiting DNA synthesis, repair and/or replication through theinhibition of dyhydrofolic acid reductase which is required forsynthesis of purine nucleotides and thymidylate. Methotrexate isindicated as a single agent or in combination with other chemotherapyagents in the treatment of choriocarcinoma, meningeal leukemia,non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovaryand bladder.

Topoisomerase I Inhibitors:

Camptothecins, including, camptothecin and camptothecin derivatives areavailable or under development as Topoisomerase I inhibitors.Camptothecins cytotoxic activity is believed to be related to itsTopoisomerase I inhibitory activity. Examples of camptothecins include,but are not limited to irinotecan, topotecan, and the various opticalforms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecindescribed below.

Irinotecan HCl, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionehydrochloride, is commercially available as the injectable solutionCAMPTOSAR®. Irinotecan is a derivative of camptothecin which binds,along with its active metabolite SN-38, to the topoisomerase I-DNAcomplex. It is believed that cytotoxicity occurs as a result ofirreparable double strand breaks caused by interaction of thetopoisomerase I:DNA:irintecan or SN-38 ternary complex with replicationenzymes. Irinotecan is indicated for treatment of metastatic cancer ofthe colon or rectum.

Topotecan HCl,(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dionemonohydrochloride, is commercially available as the injectable solutionHYCAMTIN®. Topotecan is a derivative of camptothecin which binds to thetopoisomerase I-DNA complex and prevents religation of singles strandbreaks caused by Topoisomerase I in response to torsional strain of theDNA molecule. Topotecan is indicated for second line treatment ofmetastatic carcinoma of the ovary and small cell lung cancer.

Hormones and Hormonal Analogues:

Hormones and hormonal analogues are useful compounds for treatingcancers in which there is a relationship between the hormone(s) andgrowth and/or lack of growth of the cancer. Examples of hormones andhormonal analogues useful in cancer treatment include, but are notlimited to, adrenocorticosteroids such as prednisone and prednisolonewhich are useful in the treatment of malignant lymphoma and acuteleukemia in children; aminoglutethimide and other aromatase inhibitorssuch as anastrozole, letrazole, vorazole, and exemestane useful in thetreatment of adrenocortical carcinoma and hormone dependent breastcarcinoma containing estrogen receptors; progestrins such as megestrolacetate useful in the treatment of hormone dependent breast cancer andendometrial carcinoma; estrogens, estrogens, and anti-estrogens such asfulvestrant, flutamide, nilutamide, bicalutamide, cyproterone acetateand 5α-reductases such as finasteride and dutasteride, useful in thetreatment of prostatic carcinoma and benign prostatic hypertrophy;anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, as well as selective estrogen receptor modulators (SERMS)such those described in U.S. Pat. Nos. 5,681,835, 5,877,219, and6,207,716, useful in the treatment of hormone dependent breast carcinomaand other susceptible cancers; and gonadotropin-releasing hormone (GnRH)and analogues thereof which stimulate the release of leutinizing hormone(LH) and/or follicle stimulating hormone (FSH) for the treatmentprostatic carcinoma, for instance, LHRH agonists and antagonists such asgoserelin acetate and luprolide.

Signal Transduction Pathway Inhibitors:

Signal transduction pathway inhibitors are those inhibitors, which blockor inhibit a chemical process which evokes an intracellular change. Asused herein this change is cell proliferation or differentiation. Signaltransduction inhibitors useful in the present invention includeinhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases,SH2/SH3domain blockers, serine/threonine kinases, phosphotidylinositol-3 kinases, myo-inositol signaling, and Ras oncogenes.

Several protein tyrosine kinases catalyse the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are generally termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by over-expression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2,erbB4, ret, vascular endothelial growth factor receptor (VEGFr),tyrosine kinase with immunoglobulin-like and epidermal growth factorhomology domains (TIE-2), insulin growth factor-I (IGFI) receptor,macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblastgrowth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC),ephrin (eph) receptors, and the RET protooncogene. Several inhibitors ofgrowth receptors are under development and include ligand antagonists,antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.Growth factor receptors and agents that inhibit growth factor receptorfunction are described, for instance, in Kath, John C., Exp. Opin. Ther.Patents (2000) 10(6):803-818; Shawver et al DDT Vol 2, No. 2 February1997; and Lofts, F. J. et al, “Growth factor receptors as targets”, NewMolecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr,David, CRC press 1994, London.

Tyrosine kinases, which are not growth factor receptor kinases aretermed non-receptor tyrosine kinases. Non-receptor tyrosine kinasesuseful in the present invention, which are targets or potential targetsof anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focaladhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described in Sinh, S. and Corey, S. J., (1999)Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; andBolen, J. B., Brugge, J. S., (1997) Annual review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including, PI3-K p85subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34(3) 125-32.

Inhibitors of Serine/Threonine Kinases including MAP kinase cascadeblockers which include blockers of Raf kinases (rafk), Mitogen orExtracellular Regulated Kinase (MEKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).IkB kinase family (IKKa, IKKb), PKB family kinases, akt kinase familymembers, and TGF beta receptor kinases. Such Serine/Threonine kinasesand inhibitors thereof are described in Yamamoto, T., Taya, S.,Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt,P, Samani, A., and Navab, R. (2000), Biochemical Pharmacology, 60.1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys.27:41-64; Philip, P. A., and Harris, A. L. (1995), Cancer Treatment andResearch. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal ChemistryLetters, (10), 2000, 223-226; U.S. Pat. No. 6,268,391; andMartinez-lacaci, L., et al, Int. J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful in thepresent invention. Such kinases are discussed in Abraham, R. T. (1996),Current Opinion in Immunology. 8 (3) 412-8; Canman, C. E., Lim, D. S.(1998), Oncogene 17 (25) 3301-3308; Jackson, S. P. (1997), InternationalJournal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H. etal, Cancer res, (2000) 60(6), 1541-1545.

Also useful in the present invention are Myo-inositol signalinginhibitors such as phospholipase C blockers and Myoinositol analogues.Such signal inhibitors are described in Powis, G., and Kozikowski A.,(1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workmanand David Kerr, CRC press 1994, London.

Another group of signal transduction pathway inhibitors are inhibitorsof Ras Oncogene. Such inhibitors include inhibitors offarnesyltransferase, geranyl-geranyl transferase, and CAAX proteases aswell as anti-sense oligonucleotides, ribozymes and immunotherapy. Suchinhibitors have been shown to block ras activation in cells containingwild type mutant ras, thereby acting as antiproliferation agents. Rasoncogene inhibition is discussed in Scharovsky, O. G., Rozados, V. R.,Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4)292-8; Ashby, M. N. (1998), Current Opinion in Lipidology. 9 (2) 99-102;and BioChim. Biophys. Acta, (19899) 1423(3):19-30.

As mentioned above, antibody antagonists to receptor kinase ligandbinding may also serve as signal transduction inhibitors. This group ofsignal transduction pathway inhibitors includes the use of humanizedantibodies to the extracellular ligand binding domain of receptortyrosine kinases. For example Imclone C225 EGFR specific antibody (seeGreen, M. C. et al, Monoclonal Antibody Therapy for Solid Tumors, CancerTreat. Rev., (2000), 26(4), 269-286); Herceptin® erbB2 antibody (seeTyrosine Kinase Signalling in Breast cancer:erbB Family ReceptorTyrosine Kinases, Breast cancer Res., 2000, 2(3), 176-183); and 2CBVEGFR2 specific antibody (see Brekken, R. A. et al, Selective Inhibitionof VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumorgrowth in mice, Cancer Res. (2000) 60, 5117-5124).

Anti-angiogenic Agents:

-   (i) Anti-angiogenic agents including non-receptor MEK angiogenesis    inhibitors may alo be useful. Anti-angiogenic agents such as those    which inhibit the effects of vascular edothelial growth factor, (for    example the anti-vascular endothelial cell growth factor antibody    bevacizumab [Avastin™], and compounds that work by other mechanisms    (for example linomide, inhibitors of integrin αvβ3 function,    endostatin and angiostatin);    Immunotherapeutic Agents:

Agents used in immunotherapeutic regimens may also be useful incombination with the compounds of Formula (I). Immunotherapy approaches,including for example ex-vivo and in-vivo approaches to increase theimmunogenecity of patient tumour cells, such as transfection withcytokines such as interleukin 2, interleukin 4 or granulocyte-macrophagecolony stimulating factor, approaches to decrease T-cell anergy,approaches using transfected immune cells such as cytokine-transfecteddendritic cells, approaches using cytokine-transfected tumour cell linesand approaches using anti-idiotypic antibodies

Proapoptotic Agents:

Agents used in proapoptotic regimens (e.g., bcl-2 antisenseoligonucleotides) may also be used in the combination of the presentinvention.

Cell Cycle Signalling Inhibitors

Cell cycle signalling inhibitors inhibit molecules involved in thecontrol of the cell cycle. A family of protein kinases called cyclindependent kinases (CDKs) and their interaction with a family of proteinstermed cyclins controls progression through the eukaryotic cell cycle.The coordinate activation and inactivation of different cyclin/CDKcomplexes is necessary for normal progression through the cell cycle.Several inhibitors of cell cycle signalling are under development. Forinstance, examples of cyclin dependent kinases, including CDK2, CDK4,and CDK6 and inhibitors for the same are described in, for instance,Rosania et al, Exp. Opin. Ther. Patents (2000) 10(2):215-230.

In one embodiment, the combination of the present invention comprises acompound of Formula I or a salt or solvate thereof and at least oneanti-neoplastic agent selected from anti-microtubule agents, platinumcoordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine MEK angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, and cell cycle signalinginhibitors.

In one embodiment, the combination of the present invention comprises acompound of Formula I or a salt or solvate thereof and at least oneanti-neoplastic agent which is an anti-microtubule agent selected fromditerpenoids and vinca alkaloids.

In a further embodiment, at least one anti-neoplastic agent is aditerpenoid.

In a further embodiment, at least one anti-neoplastic agent is a vincaalkaloid.

In one embodiment, the combination of the present invention comprises acompound of Formula I or a salt or solvate thereof and at least oneanti-neoplastic agent, which is a platinum coordination complex.

In a further embodiment, at least one anti-neoplastic agent ispaclitaxel, carboplatin, or vinorelbine.

In a further embodiment, at least one anti-neoplastic agent iscarboplatin.

In a further embodiment, at least one anti-neoplastic agent isvinorelbine.

In a further embodiment, at least one anti-neoplastic agent ispaclitaxel.

In one embodiment, the combination of the present invention comprises acompound of Formula I and salts or solvates thereof and at least oneanti-neoplastic agent which is a signal transduction pathway inhibitor.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a growth factor receptor kinase VEGFR2, TIE2, PDGFR, BTK,erbB2, EGFr, IGFR-1, TrkA, TrkB, TrkC, or c-fms.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a serine/threonine kinase rafk, akt, or PKC-zeta.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a non-receptor tyrosine kinase selected from the src familyof kinases.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of c-src.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of Ras oncogene selected from inhibitors of farnesyltransferase and geranylgeranyl transferase.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a serine/threonine kinase selected from the groupconsisting of PI3K.

In a further embodiment the signal transduction pathway inhibitor is adual EGFr/erbB2 inhibitor, for example N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (structure below):

In one embodiment, the combination of the present invention comprises acompound of Formula I or a salt or solvate thereof and at least oneanti-neoplastic agent which is a cell cycle signaling inhibitor.

In further embodiment, cell cycle signaling inhibitor is an inhibitor ofCDK2, CDK4 or CDK6.

Immunostimulatory Agents:

As used herein “immunostimulatory agent” refers to any agent that canstimulate the immune system. As used herein immunostimulatory agentsinclude, but are not limited to, vaccine adjuvants, such as Toll-likereceptor agonists, T-cell checkpoint blockers, such as mAbs to PD-1 andCTL4 and T-cell checkpoint agonist, such as agonist mAbs to OX-40 andICOS.

Additional examples of a further active ingredient or ingredients(anti-neoplastic agent) for use in combination or co-administered withthe presently invented compound of Formula (I) are anti-PD-L1 agents.

Anti-PD-L1 antibodies and methods of making the same are known in theart.

Such antibodies to PD-L1 may be polyclonal or monoclonal, and/orrecombinant, and/or humanized.

Exemplary PD-L1 antibodies are disclosed in:

-   -   U.S. Pat. No. 8,217,149; Ser. No. 12/633,339;    -   U.S. Pat. No. 8,383,796; Ser. No. 13/091,936;    -   U.S. Pat. No. 8,552,154; Ser. No. 13/120,406;    -   US patent publication No. 20110280877; Ser. No. 13/068,337;    -   US Patent Publication No. 20130309250; Ser. No. 13/892,671;    -   WO2013019906;    -   WO2013079174;    -   U.S. application Ser. No. 13/511,538 (filed Aug. 7, 2012), which        is the US National Phase of International Application No.        PCT/US10/58007 (filed 2010);    -   and    -   U.S. application Ser. No. 13/478,511 (filed May 23, 2012).

Additional exemplary antibodies to PD-L1 (also referred to as CD274 orB7-H1) and methods for use are disclosed in U.S. Pat. No. 7,943,743;US20130034559, WO2014055897, U.S. Pat. Nos. 8,168,179; and 7,595,048.PD-L1 antibodies are in development as immuno-modulatory agents for thetreatment of cancer.

In one embodiment, the antibody to PD-L1 is an antibody disclosed inU.S. Pat. No. 8,217,149. In another embodiment, the anti-PD-L1 antibodycomprises the CDRs of an antibody disclosed in U.S. Pat. No. 8,217,149.

In another embodiment, the antibody to PD-L1 is an antibody disclosed inU.S. application Ser. No. 13/511,538. In another embodiment, theanti-PD-L1 antibody comprises the CDRs of an antibody disclosed in U.S.application Ser. No. 13/511,538.

In another embodiment, the antibody to PD-L1 is an antibody disclosed inapplication Ser. No. 13/478,511. In another embodiment, the anti-PD-L1antibody comprises the CDRs of an antibody disclosed in U.S. applicationSer. No. 13/478,511.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (MDX-1105). Inanother embodiment, the anti-PD-L1 antibody is MPDL3280A (RG7446). Inanother embodiment, the anti-PD-L1 antibody is MEDi4736.

Additional examples of a further active ingredient or ingredients(anti-neoplastic agent) for use in combination or co-administered withthe presently invented compound of Formula (I) are PD-1 antagonist.

“PD-1 antagonist” means any chemical compound or biological moleculethat blocks binding of PD-L1 expressed on a cancer cell to PD-1expressed on an immune cell (T cell, B cell or NKT cell) and preferablyalso blocks binding of PD-L2 expressed on a cancer cell to theimmune-cell expressed PD-1. Alternative names or synonyms for PD-1 andits ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1,PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC,Btdc and CD273 for PD-L2. In any embodiments of the aspects orembodiments of the present invention in which a human individual is tobe treated, the PD-1 antagonist blocks binding of human PD-L1 to humanPD-1, and preferably blocks binding of both human PD-L1 and PD-L2 tohuman PD-1. Human PD-1 amino acid sequences can be found in NCBI LocusNo.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be foundin NCBI Locus No.: NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in the any of the aspects of the presentinvention include a monoclonal antibody (mAb), or antigen bindingfragment thereof, which specifically binds to PD-1 or PD-L1, andpreferably specifically binds to human PD-1 or human PD-L1. The mAb maybe a human antibody, a humanized antibody or a chimeric antibody, andmay include a human constant region. In some embodiments, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG3 and IgG4 constant regions, and in preferred embodiments, the humanconstant region is an IgG1 or IgG4 constant region. In some embodiments,the antigen binding fragment is selected from the group consisting ofFab, Fab′-SH, F(ab′)₂, scFv and Fv fragments.

Examples of mAbs that bind to human PD-1, and useful in the variousaspects and embodiments of the present invention, are described in U.S.Pat. Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, 8,168,757,WO2004/004771, WO2004/072286, WO2004/056875, and US2011/0271358.

Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in any ofthe aspects and embodiments of the present invention include: MK-3475, ahumanized IgG4 mAb with the structure described in WHO Drug Information,Vol. 27, No. 2, pages 161-162 (2013) and which comprises the heavy andlight chain amino acid sequences shown in FIG. 6; nivolumab, a humanIgG4 mAb with the structure described in WHO Drug Information, Vol. 27,No. 1, pages 68-69 (2013) and which comprises the heavy and light chainamino acid sequences shown in FIG. 7; the humanized antibodies h409A11,h409A16 and h409A17, which are described in WO2008/156712, and AMP-514,which is being developed by Medimmune.

Other PD-1 antagonists useful in the any of the aspects and embodimentsof the present invention include an immunoadhesin that specificallybinds to PD-1, and preferably specifically binds to human PD-1, e.g., afusion protein containing the extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region such as an Fc region of animmunoglobulin molecule. Examples of immunoadhesion molecules thatspecifically bind to PD-1 are described in WO2010/027827 andWO2011/066342. Specific fusion proteins useful as the PD-1 antagonist inthe treatment method, medicaments and uses of the present inventioninclude AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusionprotein and binds to human PD-1.

Other examples of mAbs that bind to human PD-L1, and useful in thetreatment method, medicaments and uses of the present invention, aredescribed in WO2013/019906, WO2010/077634 A1 and U.S. Pat. No.8,383,796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonistin the treatment method, medicaments and uses of the present inventioninclude MPDL3280A, BMS-936559, MEDi4736, MSB0010718C.

KEYTRUDA/pembrolizumab is an anti-PD-1 antibody marketed for thetreatment of lung cancer by Merck. The amino acid sequence ofpembrolizumab and methods of using are disclosed in U.S. Pat. No.8,168,757.

Opdivo/nivolumab is a fully human monoclonal antibody marketed byBristol Myers Squibb directed against the negative immunoregulatoryhuman cell surface receptor PD-1 (programmed death-1 or programmed celldeath-1/PCD-1) with immunopotentiation activity. Nivolumab binds to andblocks the activation of PD-1, an Ig superfamily transmembrane protein,by its ligands PD-L1 and PD-L2, resulting in the activation of T-cellsand cell-mediated immune responses against tumor cells or pathogens.Activated PD-1 negatively regulates T-cell activation and effectorfunction through the suppression of PI3k/Akt pathway activation. Othernames for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. Theamino acid sequence for nivolumab and methods of using and making aredisclosed in U.S. Pat. No. 8,008,449.

Additional examples of a further active ingredient or ingredients(anti-neoplastic agent) for use in combination or co-administered withthe presently invented compound of Formula (I) are immuno-modulators.

As used herein “immuno-modulators” refer to any substance includingmonoclonal antibodies that affects the immune system. The ICOS bindingproteins of the present invention can be considered immune-modulators.Immuno-modulators can be used as anti-neoplastic agents for thetreatment of cancer. For example, immune-modulators include, but are notlimited to, anti-CTLA-4 antibodies such as ipilimumab (YERVOY) andanti-PD-1 antibodies (Opdivo/nivolumab and Keytruda/pembrolizumab).Other immuno-modulators include, but are not limited to, OX-40antibodies, PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, 41BBantibodies and GITR antibodies.

Yervoy (ipilimumab) is a fully human CTLA-4 antibody marketed by BristolMyers Squibb. The protein structure of ipilimumab and methods are usingare described in U.S. Pat. Nos. 6,984,720 and 7,605,238.

CD134, also known as OX40, is a member of the TNFR-superfamily ofreceptors which is not constitutively expressed on resting naïve Tcells, unlike CD28. OX40 is a secondary costimulatory molecule,expressed after 24 to 72 hours following activation; its ligand, OX40L,is also not expressed on resting antigen presenting cells, but isfollowing their activation. Expression of OX40 is dependent on fullactivation of the T cell; without CD28, expression of OX40 is delayedand of fourfold lower levels. OX-40 antibodies, OX-40 fusion proteinsand methods of using them are disclosed in U.S. Pat. Nos: 7,504,101;7,758,852; 7,858,765; 7,550,140; 7,960,515; WO2012027328; WO2013028231.

The term “Toll-like receptor” (or “TLR”) as used herein refers to amember of the Toll-like receptor family of proteins or a fragmentthereof that senses a microbial product and/or initiates an adaptiveimmune response. In one embodiment, a TLR activates a dendritic cell(DC). Toll-like receptors (TLRs) are a family of pattern recognitionreceptors that were initially identified as sensors of the innate immunesystem that recognize microbial pathogens. TLRs recognize distinctstructures in microbes, often referred to as “PAMPs” (pathogenassociated molecular patterns). Ligand binding to TLRs invokes a cascadeof intra-cellular signaling pathways that induce the production offactors involved in inflammation and immunity. In humans, ten TLR havebeen identified. TLRs that are expressed on the surface of cells includeTLR-1, -2, -4, -5, and -6, while TLR-3, -7/8, and -9 are expressed withthe ER compartment. Human DC subsets can be identified on the basis ofdistinct TLR expression patterns. By way of example, the myeloid or“conventional” subset of DC (mDC) expresses TLRs 1-8 when stimulated,and a cascade of activation markers (e.g. CD80, CD86, MHC class I andII, CCR7), pro-inflammatory cytokines, and chemokines are produced. Aresult of this stimulation and resulting expression is antigen-specificCD4+ and CD8+ T cell priming. These DCs acquire an enhanced capacity totake up antigens and present them in an appropriate form to T cells. Incontrast, the plasmacytoid subset of DC (pDC) expresses only TLR7 andTLR9 upon activation, with a resulting activation of NK cells as well asT-cells. As dying tumor cells may adversely affect DC function, it hasbeen suggested that activating DC with TLR agonists may be beneficialfor priming anti-tumor immunity in an immunotherapy approach to thetreatment of cancer. It has also been suggested that successfultreatment of breast cancer using radiation and chemotherapy requiresTLR4 activation.

TLR agonists known in the art and finding use in the present inventioninclude, but are not limited to, the following: Pam3Cys, a TLR1/2agonist; CFA, a TLR2 agonist; MALP2, a TLR2 agonist; Pam2Cys, a TLR2agonist; FSL-I, a TLR-2 agonist; Hib-OMPC, a TLR-2 agonist;polyribosinic:polyribocytidic acid (Poly I:C), a TLR3 agonist;polyadenosine-polyuridylic acid (poly AU), a TLR3 agonist;Polyinosinic-Polycytidylic acid stabilized with poly-L-lysine andcarboxymethylcellulose (Hiltonol), a TLR3 agonist; bacterial flagellin aTLR5 agonist; imiquimod, a TLR7 agonist; resiquimod, a TLR7/8 agonist;loxoribine, a TLR7/8 agonist; and unmethylated CpG dinucleotide(CpG-ODN), a TLR9 agonist.

Additional TLR agonists known in the art and finding use in the presentinvention further include, but are not limited to aminoalkylglucosaminide phosphates (AGPs) which bind to the TLR4 receptor areknown to be useful as vaccine adjuvants and immunostimulatory agents forstimulating cytokine production, activating macrophages, promotinginnate immune response, and augmenting antibody production in immunizedanimals. An example of a naturally occurring TLR4 agonist is bacterialLPS. An example of a semisynthetic TLR4 agonist is monophosphoryl lipidA (MPL). AGPs and their immunomodulating effects via TLR4 are disclosedin patent publications such as WO 2006/016997, WO 2001/090129, and/orU.S. Pat. No. 6,113,918 and have been reported in the literature.Additional AGP derivatives are disclosed in U.S. Pat. Nos. 7,129,219,6,525,028 and 6,911,434. Certain AGPs act as agonists of TLR4, whileothers are recognized as TLR4 antagonist.

In addition to the immunostimulatory agents described above, thecompositions of the present invention may further comprise one or moreadditional substances which, because of their adjuvant nature, can actto stimulate the immune system to respond to the cancer antigens presenton the inactivated tumor cell(s). Such adjuvants include, but are notlimited to, lipids, liposomes, inactivated bacteria which induce innateimmunity (e.g., inactivated or attenuated I/ster/a monocytogenes),compositions which mediate innate immune activation via, (NOD)-likereceptors (NLRs), Retinoic acid inducible gene-based (RIG)-l-likereceptors (RLRs), and/or C-type lectin receptors (CLRs). Examples ofPAMPs include lipoproteins, lipopolypeptides, peptidoglycans, zymosan,lipopolysaccharide, neisserial porins, flagellin, profillin,galactoceramide, muramyl dipeptide. Peptidoglycans, lipoproteins, andlipoteichoic acids are cell wall components of Gram-positive.Lipopolysaccharides are expressed by most bacteria, with MPL being oneexample. Flagellin refers to the structural component of bacterialflagella that is secreted by pathogenic and commensal bacterial.rt.-Galactosylceramide (rt.-GalCer) is an activator of natural killer T(NKT) cells. Muramyl dipeptide is a bioactive peptidoglycan motif commonto all bacteria.

Because of their adjuvant qualities, TLR agonists are preferably used incombinations with other vaccines, adjuvants and/or immune modulators,and may be combined in various combinations. Thus, in certainembodiments, the herein described compounds of Formula (I) that bind toSTING and induce STING-dependent TBKI activation and an inactivatedtumor cell which expresses and secretes one or more cytokines whichstimulate DC induction, recruitment and/or maturation, as describedherein can be administered together with one or more TLR agonists fortherapeutic purposes.

Additional examples of a further active ingredient or ingredients(anti-neoplastic agent) for use in combination or co-administered withthe presently invented compound of Formula (I) are antibodies to ICOS.

CDRs for murine antibodies to human ICOS having agonist activity areshown in PCT/EP2012/055735 (WO 2012/131004). Antibodies to ICOS are alsodisclosed in WO 2008/137915, WO 2010/056804, EP 1374902, EPI374901, andEP1125585.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key immunosuppressive enzymethat modulates the anti-tumor immune response by promoting regulatory Tcell generation and blocking effector T cell activation, therebyfacilitating tumor growth by allowing cancer cells to avoid immunesurveillance. (Lemos H, et al., Cancer Res. 2016 Apr. 15;76(8):2076-81), (Munn D H, et al., Trends Immunol. 2016 March;37(3):193-207). Further active ingredients (anti-neoplastic agents) foruse in combination or co-administered with the presently inventedcompounds of Formula (I) are IDO inhibitors. Epacadostat,((Z)—N-(3-bromo-4-fluorophenyl)-N′-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazole-3-carboxamidine)is a highly potent and selective oral inhibitor of the IDO1 enzyme thatreverses tumor-associated immune suppression and restores effectiveanti-tumor immune responses. Epacadostat is disclosed in U.S. Pat. No.8,034,953.

Additional examples of a further active ingredient or ingredients(anti-neoplastic agent) for use in combination or co-administered withthe presently invented compound of Formula (I) are CD73 inhibitors andA2a and A2b adenosine antagonists.

In one aspect the disease to be treated is an infectious disease, egcaused by bacteria or virus.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof for use in thetreatment of infectious disease.

In a further aspect there is provided a method of treating infectiousdisease comprisingadministering to a human in need thereof atherapeutically effective amount of a compoundof Formula (I) or apharmaceutically acceptable salt thereof.

In a further aspect there is provided the use of a compound of Formula(I) or a pharmaceutically acceptable salt thereof in the manufacture ofa medicament for the treatment of infectious disease.

In one embodiment, the compound of the invention may be employed withother therapeutic methods of treating infectious disease. In particular,antiviral and antibacterial agents are envisaged.

The compounds of Formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more agents useful in theprevention or treatment of bacterial and viral infections. Examples ofsuch agents include, without limitation; polymerase inhibitors such asthose disclosed in WO 2004/037818-A1, as well as those disclosed in WO2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796,R-803, R1728, R1626, as well as those disclosed in WO 2006/018725, WO2004/074270, WO 2003/095441, US2005/0176701, WO 2006/020082, WO2005/080388, WO 2004/064925, WO 2004/065367, WO 2003/007945, WO02/04425, WO 2005/014543, WO 2003/000254, EP 1065213, WO 01/47883, WO2002/057287, WO 2002/057245 and similar agents; replication inhibitorssuch as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine andsimilar agents; protease inhibitors such as the HIV protease inhibitorssaquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir,brecanavir, atazanavir, tipranavir, palinavir, lasinavir, and the HCVprotease inhibitors BILN2061, VX-950, SCH₅₀₃₀₃₄; and similar agents;nucleoside and nucleotide reverse transcriptase inhibitors such aszidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavidine,adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine,alovudine, amdoxovir, elvucitabine, and similar agents; non-nucleosidereverse transcriptase inhibitors (including an agent havinganti-oxidation activity such as immunocal, oltipraz etc.) such asnevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, TMC-278, TMC-125, etravirine, and similar agents; entryinhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140,TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors suchas L-870,180 and similar agents; budding inhibitors such as PA-344 andPA-457, and similar agents; chemokine receptor inhibitors such asvicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, aswell as those disclosed in WO 02/74769, WO 2004/054974, WO 2004/055012,WO 2004/055010, WO 2004/055016, WO 2004/055011, and WO 2004/054581, andsimilar agents; neuraminidase inhibitors such as CS-8958, zanamivir,oseltamivir, peramivir and similar agents; ion channel blockers such asamantadine or rimantadine and similar agents; and interfering RNA andantisense oligonucleotides and such as ISIS-14803 and similar agents;antiviral agents of undetermined mechanism of action, for example thosedisclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin,and similar agents. The compounds of Formula (I) and pharmaceuticallyacceptable salts thereof may also be used in combination with one ormore other agents which may be useful in the prevention or treatment ofviral infections for example immune therapies (e.g. interferon or othercytokines/chemokines, cytokine/chemokine receptor modulators, cytokineagonists or antagonists and similar agents); and therapeutic vaccines,antifibrotic agents, anti-inflammatory agents such as corticosteroids orNSAIDs (non-steroidal anti-inflammatory agents) and similar agents.

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent useful in the treatment ofinfectious disease

In a further aspect there is provided a combination comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand at least one further therapeutic agent useful in the treatment ofinfectious diseasefor use in therapy.

In a further aspect there is provided a combination comprising acompound of Formula (I) or pharmaceutically acceptable salt thereof andat least one one further therapeutic agent useful in the treatment ofinfectious disease, for use in the treatment of infectious disease.

In a further aspect there is provided the use of a combinationcomprising a compound of Formula (I) or a pharmaceutically acceptablesalt thereof and at least one one further therapeutic agent useful inthe treatment of infectious diseasein the manufacture of a medicamentfor the treatment of infectious disease.

In a further aspect there is provided a method of treating infectiousdisease comprising administering to a human in need thereof atherapeutically effective amount of a combination comprising a compoundof Formula (I) or a pharmaceutically acceptable salt thereof and atleast one further therapeutic agent useful in the treatment ofinfectious disease.

In a further aspect there is provided a pharmaceutical compositioncomprising a combination comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof and at least one furthertherapeutic agent useful in the treatment of infectious disease and oneor more of pharmaceutically acceptable excipients.

In a further aspect there is provided a composition comprising acompound of Formula (I), or a pharmaceutically acceptable salt thereof,and one or more immunostimulatory agents.

There is also therefore provided an immugenic composition or vaccineadjuvant comprising a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided an immugenic composition comprising an antigenor antigen composition and a compound of Formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a vaccine composition comprising an antigen orantigen composition and a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, an immugenic composition comprising an antigenor antigen composition and a compound of Formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, a vaccine composition comprising an antigen orantigen composition and a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided the use of a compound of Formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of animmugenic composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

There is further provided the use of a compound of Formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of avaccine composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

It will be appreciated that the compounds depicted in the applicationmay be drawn using different conventions. For example, the following twocompounds are considered equivalent in chemical structure andstereochemistry.

COMPOUND PREPARATION and EXAMPLES

Compounds of Formula (I), where Y¹, Y², X¹, X², R¹, R², R³, R⁴, R⁵, R⁶,R⁸, and R⁹ are as defined hereinbefore, may be prepared by methods knownin the art of organic synthesis as set forth in the schemes and Examplesbelow. In all of the methods, it is well understood that protectinggroups for sensitive or reactive groups may be employed where necessaryin accordance with general principles of chemistry. Protecting groupsare manipulated according to standard methods of organic synthesis (P.G. M. Wuts and T. W. Green (2007) Greene's Protective Groups in OrganicSynthesis, 4^(th) edition, John Wiley & Sons). These groups are removedat a convenient stage of the compound synthesis using methods that arereadily apparent to those skilled in the art. The selection of processesas well as the reaction conditions and order of their execution shall beconsistent with the preparation of compounds of Formula (I).

The compounds of Formula (I) and salts thereof may be prepared by themethodology described hereinafter, constituting further aspects of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of Formula (I), in which R⁵ is OC(O)R⁷ and R⁶ is F, and both Y¹and Y² are O, both X¹ and X² are S⁻, as illustrated as formula (IIa),wherein R¹, R², R³ and R⁴ are as defined hereinbefore for a compound ofFormula (I). The process comprises the acylation of a compound offormula (IIIa):

wherein R¹, R², R³, R⁴ and R⁷ are as defined hereinbefore for a compoundof formula (IIa) and thereafter, if required, preparing a salt of thecompound so-formed.

Example 1

A compound of formula (IIIa) and myristic anyhydride in a suitablesolvent, for example dimethylformamide (DMF), in the presence of a basesuch as pyridine, is stirred at room temperature or heated at a suitabletemperature, for example 60° C., for a suitable period of time, forexample 2-48 hours. The product, of formula (IIa), is isolated byremoval of the volatiles and purification if required.

There is also provided a process for the preparation of a compound ofFormula (I), in which R⁵ is OH and R⁶ is F, and both Y¹ and Y² are O,both X¹ and X² are S, and both R⁸ and R⁹ are CH₂OC(O)tBu, as illustratedas formula (IVa), wherein R¹, R², R³ and R⁴ are as defined hereinbeforefor a compound of Formula (I). The process comprises the addition of ancarbonyloxymethyl group to a compound of formula (IIIa):

Example 2

A compound of formula (IIIa) and chloromethyl pivalate (POM-CI) in asuitable solvent, for example dimethylformamide (DMF), in the presenceof a base such as Et₃N, is stirred at room temperature for a suitableperiod of time, for example 48 hours. The product, of formula (IVa), isisolated by removal of the volatiles and purification if required.

A compound of formula (IIIa) may be prepared by deprotection of acompound of formula (Va):

wherein R¹, R², R³ and R⁴ are as defined hereinbefore for a compound offormula (IIIa) and P¹ is a suitable protecting group, such as,tert-butyldimethylsilyloxy (TBDMS) and thereafter, if required,preparing a salt of the compound so-formed.

Example 3

A compound of formula (Va), in a suitable solvent, for example pyridineis heated at a suitable temperature, for example 50° C., then treatedwith a mixture of triethylamine trihydrofluoride and triethylamine, fora suitable period of time, for example 2-3 hours. The product, offormula (IIIa), is isolated by precipitation by the addition of asolvent, for example acetone, or by removal of the volatiles, andpurification if required.

A compound of formula (Va) may be prepared by deprotection of a compoundof formula (VIa):

wherein, P¹ is a protecting group as defined for compound of formula(Va) and R¹², R¹³, R¹⁴ and R¹⁵ are defined as

-   R¹² is OH and R¹³ is NHCOiPr, or R¹² is NHBz and R¹³ is H;-   R¹⁴ is OH and R¹⁵ is NHCOiPr or R¹⁴ is NHBz and R¹⁵ is H;

Example 4

A compound of formula (VIa) is dissolved in a suitable mixture, forexample, methylamine in methanol or aqueous ammonia in methanol, andheated at a suitable temperature, for example 50-55° C., for a suitableperiod of time, for example 2-72 hours. The product, of formula (Va), isisolated by removal of the solvent and purification if required.

A compound of formula (VIa) may be prepared by reaction of a compound offormula (VIIa):

wherein, P¹, R¹², R¹³, R¹⁴ and R¹⁵ are defined as hereinbefore for acompound of formula (VIa).

Example 5

A compound of formula (VIIa) is dissolved in a suitable solvent, forexample, pyridine, and treated with a suitable coupling reagent, forexample, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, andstirred at a suitable temperature, for example 20° C., for a suitableperiod of time, for example 0.5-2 hours. Quenching of the reaction byaddition of a suitable solvent, for example water, then after theaddition of an sulfurizing agent, for example3H-benzo[c][1,2]dithiol-3-one, and stirring at a suitable temperature,for example 20° C., for a suitable period of time, for example 5-10minutes. Quenching of the reaction by addition of a suitable solvent,for example aqueous NaHCO₃ solution. Extraction of the product, offormula (VIa), by a suitable organic solvent such as EtOAc. The product,of formula (VIa), is isolated by removal of the solvent and purificationif required.

A compound of formula (VIIa) may be prepared by reaction of a compoundof formula (VIIIa) with a compound of formula (IXa):

wherein, P¹, R¹², R¹³, R¹⁴ and R¹⁵ are defined as hereinbefore for acompound of formula (VIIa) and DMTr is a 4,4-dimethoxytrityl protectinggroup.

Example 6

A compound of formula (IXa) in a suitable solvent, for example,acetonitrile in the presence of molecular sieves, is treated with asolution of a compound of formula (VIIIa) dissolved in a suitablesolvent, for example acetonitrile, and stirred at a suitabletemperature, for example 20° C., for a suitable period of time, forexample 0.5-2 hours. A solution of a suitable sulfurizing agent, forexample N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(DDTT), is added and the mixture is stirred at a suitable temperature,for example 20° C., for a suitable period of time, for example 0.5-1hour. After evaporation of the solvent, the residue is dissolved in asuitable solvent, for example a mixture of dichloromethane and water,and treated with a suitable reagent, for example dichloroacetic acid,and stirred at a suitable temperature, for example 20° C., for asuitable period of time, for example 15 minutes. A solution containingthe product, of formula (VIIa), is obtained by the addition of asuitable solvent, for example pyridine, and concentration byevaporation.

A compound of formula (VIIIa) may be prepared by reaction of a compoundof formula (Xa).

wherein, R¹² and R¹³ are defined as hereinbefore for a compound offormula (VIIIa) and DMTr is a 4,4-dimethoxytrityl protecting group.

Example 7

A compound of formula (Xa) is dissolved in a suitable mixture, forexample, acetonitrile containing water, is treated with pyridiniumtrifluoroacetate, and stirred at a suitable temperature, for example 20°C., for a suitable period of time, for example 1-5 minutes. Thentert-butylamine is added and the mixture is stirred at a suitabletemperature, for example 20° C., for a suitable period of time, forexample 10 minutes. The product is isolated by evaporation of thesolvent then dissolved in a suitable solvent, for exampledichloromethane containing water, and treated with dichloroacetic acidand stirred at a suitable temperature, for example 20° C., for asuitable period of time, for example 15 minutes. A concentrated solutionof the product, of formula (VIIIa), in acetonitrile is obtained by theaddition of pyridine followed by azeotroping the mixture with anhydrousacetonitrile.

Phosphoramidites of formula (IXa) and (Xa) are either known in theliterature, or are commercially available from suppliers such as Sigma,Chemgenes and CarboSynth or may be prepared by known methods.

Example 8 Compound 1b(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

Intermediate 1(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (2190 mg, 2.5 mmol) inacetonitrile (15 mL) and water (0.090 mL, 5.00 mmol) at room temperaturewas added pyridine 2,2,2-trifluoroacetate (579 mg, 3.00 mmol). Themixture was stirred for 1 minute, after which time LCMS indicatedcomplete conversion to the first intermediate, m/z (M+H)=793.3. Then,2-methylpropan-2-amine (13.14 mL, 125 mmol) was added and the mixturestirred for 10 minutes, after which time LCMS indicated consumption ofthe first formed intermediate.

The mixture was concentrated in vacuo to afford a white foam. The foamwas then dissolved in acetonitrile (20 mL) and concentrated. Thisprocess was repeated one more time. The crude material was dissolved indichloromethane (10 mL) and purified by chromatography in two batches(silica gel, gradient elution of 0-30% methanol in dichloromethane). Thedesired fractions were combined and evaporated to afford two separatewhite solids which were then dissolved in dichloromethane, combined andevaporated to afford the titled compound (780 mg, 1.055 mmol, 42.2%yield) as a white solid. LCMS m/z 740.4 (M+H).

Intermediate 2(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

Intermediate 2 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 2 inother Examples, may be used.

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (775 mg, 1.048 mmol) in dichloromethane (20 mL) andwater (0.189 mL, 10.48 mmol) at room temperature was added2,2-dichloroacetic acid (0.655 mL, 8.38 mmol). The mixture was stirredat room temperature for 30 minutes.

The reaction was then quenched by addition of pyridine (1.356 mL, 16.76mmol) and concentrated in vacuo to afford a colorless oil. The materialwas stored under nitrogen at 4° C. After storing at 4° C., the materialsolidified to afford the impure titled compound as a waxy, white solid,which was used without further purification. LCMS m/z 438.3 (M+H).

Intermediate 3(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate

Intermediate 3 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 3 inother Examples, may be used.

The impure solid(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (458 mg, 1.047 mmol) obtained above was azeotropedwith anhydrous acetonitrile (3×20 mL). After the last concentration, ˜10mL of acetonitrile was kept in the flask. At room temperature,(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (1345 mg, 1.361 mmol) wasdried by azeotroping with anhydrous acetonitrile (3×20 mL). After thelast concentration, ˜5 mL of acetonitrile was kept in the flask, and 3 Åmolecular sieves (˜40 beads) were added. The solution was left standingover the molecular sieves at room temperature for 1 hour.

To the dried suspension of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (458 mg, 1.047 mmol) in acetonitrile (10 mL) atroom temperature under a nitrogen atmosphere was added the pre-driedsolution of(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (1345 mg, 1.361 mmol) inacetonitrile (5 mL) via syringe. The solution went from being orange tolight yellow. The mixture was stirred under a nitrogen atmosphere atroom temperature for 1 hour, after which time LCMS indicated minimalremaining starting material. Then,(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (237mg, 1.152 mmol) was added to the reaction mixture and the mixture wasstirred at room temperature for 30 minutes. The solvent was evaporatedin vacuo, and the residue was taken into dichloromethane (40 mL) andwater (0.189 mL, 10.47 mmol), followed by the addition of2,2-dichloroacetic acid (1.037 mL, 12.57 mmol). The mixture was stirredat room temperature for 10 minutes, after which time LCMS indicated theformation of desired product. The reaction was quenched with pyridine(10 mL, 124 mmol), then the mixture was concentrated in vacuo to affordthe impure titled compound as an orange oil. LCMS: m/z 1054 (M+H). Theproduct was stored under nitrogen at 4° C. and used in the followingstep without further purification.

Intermediate 4N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide

A solution of crude(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogenphosphonate (1104 mg, 1.047 mmol) in pyridine (˜20 mL) and evaporated,then redissolved in pyridine (20 mL) and concentrated to approximately10 mL. To this solution under nitrogen was added2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (677 mg, 3.67 mmol)in one portion. The reaction mixture was stirred at room temperatureunder nitrogen for 30 minutes, after which time LCMS indicatedconsumption of starting material, then the reaction was quenched by theaddition of water (0.660 mL, 36.7 mmol). 3H-Benzo[c][1,2]dithiol-3-one(264 mg, 1.571 mmol) was then added, and the mixture stirred at roomtemperature for 5 minutes before being poured onto a solution of water(160 mL) containing sodium bicarbonate (4400 mg, 52.4 mmol). Thismixture was stirred for 5 minutes, then EtOAc (150 mL) was added, andthe mixture stirred for a further 10 minutes. The solution wastransferred to a separating funnel, and the aqueous layer was separatedfrom the organic. The aqueous layer was further extracted with EtOAc(150 mL), then the combined organic layers were dried (Na₂SO₄), filteredand evaporated in vacuo to an orange oil. The material was stored undernitrogen at 4° C. overnight.

After warming to room temperature, the crude material was diluted withtoluene (20 mL) and evaporated to remove excess pyridine. The crudeproduct was purified by chromatography (silica gel, ISCO Teledyne Gold,80 g) Gradient elution was run from 0-10% methanol in dichloromethaneover 15 minutes followed by a 5 minute isocratic hold at 10% methanol indichloromethane. Then the gradient increased from 10-20% methanol indichloromethane over 15 minutes followed by a 10 minute isocratic holdat 20% methanol in dichloromethane. The desired fractions were combinedand evaporated in vacuo to afford the titled compound (570 mg, 0.342mmol, 32.6% yield) as a yellow solid.

Four isomers were observed by LCMS [m/z 1068.5 (M+H)] in an approximateratio of 8:4:2:1 with retention times of 1.13, 1.23, 1.18 and 1.08minutes, respectively. The product was stored under nitrogen at 4° C.and used in the following step without further purification.

Intermediate 5(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]l-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

A solution of above obtainedN-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(570 mg, 0.534 mmol) in methylamine (33 wt % in ethanol) (25 mL, 201mmol) was stirred at room temperature for 50 minutes, after which timeLCMS indicated consumption of starting materials. The reaction mixturewas concentrated in vacuo to an orange residue. The material was storedunder nitrogen at 4° C. overnight.

The material was allowed to warm to room temperature and dissolved inmethanol/DMSO (4 mL total). A portion was purified using reverse phaseHPLC (10-90% acetonitrile:water (with 0.1% NH₄OH modifier), C18 50×30 mmGemini column, 47 mL/min, 8 minute gradient, UV collection=214 nm).

Another portion was purified using reverse phase HPLC (10-50%acetonitrile:water (with 0.1% NH₄OH modifier), C18 50×30 mm Geminicolumn, 47 mL/min, 8 minute gradient, UV collection=214 nm).

Fractions from the two purifications were combined and concentrated toafford three isomeric products:

-   -   Isomer 1 of the titled compound as a bisammonium salt, with the        exact stereochemistry at two phosphorus centers undetermined (6        mg, purity by LCMS =70%; 4.99 μmol, 0.936% yield) as an        off-white gum, LCMS m/z 807.2 (M+H), t_(RET)=0.68 min.    -   Isomer 2 of the titled compound as a bisammonium salt, with the        exact stereochemistry at two phosphorus centers undetermined (64        mg, purity by LCMS =22%, 0.017 mmol, 3.14% yield) as a colorless        gum, LCMS m/z 807.2 (M+H), tRET=0.80 min.    -   Isomer 3 of the titled compound as a bisammonium salt, with the        exact stereochemistry at two phosphorus centers undetermined (26        mg, purity by LCMS =50%, 0.015 mmol, 2.90% yield) as a white        solid, LCMS m/z 807.2 (M+H), t_(RET)=0.92 min.

The latest eluting isomer, Isomer 3 of the titled compound, was themajor product as determined by area under the chromatography peak (UV @214 nm) and was used in the following deprotection step.

Example 8(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

Isomer 3 of(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(25 mg, 0.031 mmol) obtained from the preceding step was suspended inpyridine (0.5 mL) and triethylamine (0.5 mL). The mixture was stirredand heated to 50° C., then triethylamine trihydrofluoride (0.5 mL, 3.07mmol) was added and the mixture was stirred at 50° C. for a further 2hours by which time LCMS indicated the complete consumption of startingmaterial and conversion to the desired product.

The mixture was allowed to cool to room temperature, then acetone (˜10mL) was added and the solvent evaporated and the material stored undernitrogen at 4° C. overnight.

The crude residue was purified using reverse phase HPLC (0-20%acetonitrile in water (0.1% NH₄OH), 50×30 mm Gemini column, 47 mL/min, 8minute gradient, UV detection @ 214 nm). The desired fractions werecombined and evaporated to afford the titled compound as a bisammoniumsalt (2.3 mg) as a single diastereomer, with the exact stereochemistryat two phosphorus centers undetermined. The product was a white solid.LCMS m/z 693.1 (M+H).

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.69 (s, 1H), 8.33(s, 1H), 8.14-8.17 (m, 1H), 8.12 (br s, 1H), 6.24 (br dd, J=14.5, 3.2Hz, 1H), 6.08 (br d, J=8.3 Hz, 1H), 5.71-5.86 (m, 1H), 5.22 (br t, J=8.7Hz, 2H), 4.49-4.54 (m, 1H), 4.32 (br s, 1H), 4.16 (br s, 1H), 4.08-4.15(m, 2H), 4.03-4.06 (m, 1H), 4.00-4.03 (m, 1H), 3.73 (br s, 1H).

¹³C NMR (150 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 156.1, 155.9,153.2, 152.9, 150.4, 149.1, 119.1, 118.4, 90.6, 85.3, 83.6, 83.0, 80.6,77.7, 71.6, 71.1, 67.2, 63.3.

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 53.84 and 49.04.

It is noted that Example 8 produced Compound 1b-Isomer 2. Because of thesmall scale of the reaction, no Compound 1a-Isomer 1 was isolated. BothIsomer 1 and Isomer 2 of Compound 1 are prepared in Examples 8a and 8bbelow.

Examples 8a and 8b Compounds 1a and 1b(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

Intermediate 1(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (10 g, 11.42 mmol) inacetonitrile (65 mL) and water (0.411 mL, 22.83 mmol) at roomtemperature was added pyridine 2,2,2-trifluoroacetate (2.65 g, 13.70mmol). The mixture was stirred for 1 minute, after which time LCMSindicated complete conversion to the first intermediate m/z 793.7 (M+H).Then, 2-methylpropan-2-amine (60.0 mL, 571 mmol) was added and themixture stirred for 15 minutes, after which time LCMS indicatedconsumption of the first formed intermediate.

The mixture was concentrated in vacuo to a white foam. The foam was thendissolved in acetonitrile and concentrated (50 mL). This process wasrepeated one more time. The crude material was dissolved indichloromethane and purified by chromatography (silica gel, ISCORediSep, 120 g silica) eluted with a gradient of 0-30% methanol indichloromethane. The desired fractions were combined and evaporated invacuo to afford the titled compound (4.8 g, 6.49 mmol, 56.8% yield) as awhite solid. LCMS m/z 740.3 (M+H).

Intermediate 2(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

Intermediate 2 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 2 inother Examples, may be used.

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (4.8 g, 6.49 mmol) in dichloromethane (100 mL) andwater (1.169 mL, 64.9 mmol) at room temperature was added2,2-dichloroacetic acid (4.06 mL, 51.9 mmol). The mixture was stirred atroom temperature for 30 minutes. The reaction was then quenched withpyridine (8.40 mL, 104 mmol) and concentrated in vacuo to afford theimpure titled compound as a colorless oil. The material was used as isimmediately in the next step. A final mass was not determined. LCMS m/z438.3 (M+H).

Intermediate 3(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogenphosphonate

Intermediate 3 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 2 inother Examples, may be used.

The impure solid(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (2.84 g, 6.49 mmol) obtained above was azeotropedwith anhydrous acetonitrile (3×60 mL). After the last concentration, ˜20mL of acetonitrile was kept in the flask. At room temperature,(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (8.00 g, 8.10 mmol) was driedby azeotroping with anhydrous acetonitrile (3×60 mL). After the lastconcentration, ˜30 mL of acetonitrile was kept in the flask, and 3 Amolecular sieves (˜40 beads) were added. The solution was left standingover the molecular sieves at room temperature for 1 hour.

To the dried suspension of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (2.84 g, 6.49 mmol) in acetonitrile (40 mL) at roomtemperature under a nitrogen atmosphere was added the pre-dried solutionof(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (8.00 g, 8.10 mmol) inacetonitrile (30 mL) via syringe. The mixture was stirred under anitrogen atmosphere at room temperature for 1 hour, after which timeLCMS indicated minimal remaining starting material. Then,(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(1.467 g, 7.14 mmol) was added to the reaction mixture, and the mixturewas stirred at room temperature for 45 minutes. The solvent wasevaporated in vacuo, and the residue was taken into dichloromethane (100mL) and water (1.170 mL, 64.9 mmol), followed by the addition of2,2-dichloroacetic acid (6.43 mL, 78 mmol). The mixture was stirred atroom temperature for 10 minutes, after which time LCMS indicated theformation of desired product. The reaction was quenched with pyridine(12.61 mL, 156 mmol), then the mixture was concentrated in vacuo toafford the impure titled compound as an orange oil. LCMS indicated theformation of two isomers with two overlaping peaks between 0.98-1.04min. The material was immediately taken on in the next step as is. Afinal sample mass was not determined. LCMS m/z 1054.6 (M+H).

Intermediate 4N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide

A solution of crude(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogenphosphonate (6.84 g, 6.49 mmol) was azeotroped from pyridine (3×50 mL),leaving pyridine (40 mL) after the last concentration. To this solutionunder nitrogen was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane2-oxide (4.19 g, 22.71 mmol) in one portion. The reaction stirred atroom temperature under nitrogen for 30 minutes, after which time LCMSindicated consumption of starting material, then the reaction wasquenched with the addition of water (4.09 mL, 227 mmol). Then,3H-benzo[c][1,2]dithiol-3-one (1.638 g, 9.73 mmol), and the mixturestirred at room temperature for 5 minutes before being poured onto asolution of water (400 mL) containing sodium bicarbonate (27.3 g, 324mmol). The mixture was stirred for 10 minutes, then EtOAc (200 mL) wasadded, and the mixture stirred for a further 10 minutes. The solutionwas transferred to a separatory funnel, and the aqueous layer wasseparated from the organic. The aqueous layer was further extracted withEtOAc (2×200 mL), then the combined organic layers were dried (Na₂SO₄),filtered and evaporated in vacuo to an orange oil. The material wasstored under nitrogen at 4° C. until use in the following step.

After warming to room temperature, the crude material was diluted withtoluene (50 mL) and evaporated to remove excess pyridine. This processwas repeated two more times. The crude product was purified bychromatography (silica gel, ISCO Teledyne Gold, 220 g silica). Gradientelution was run from 0-10% methanol in dichloromethane over 15 minutesfollowed by a 5 minute isocratic hold at 10% methanol indichloromethane. Then the gradient increased from 10-20% methanol indichloromethane over 15 minutes followed by a 5 minute isocratic hold at20% methanol in dichloromethane. The desired fractions were combined andevaporated in vacuo to afford an orange oil. The material was storedunder nitrogen at 4° C. overnight.

After warming to room temperature, the material was purified again bychromatography (silica gel, ISCO Teledyne Gold, 120 g silica). Gradientelution was run from 0-10% methanol in dichloromethane over 15 minutesfollowed by a 5 minute isocratic hold at 10% methanol indichloromethane. Then the gradient increased from 10-20% methanol indichloromethane over 10 minutes followed by a 5 minute isocratic hold at20% methanol in dichloromethane. The desired fractions were combined andevaporated in vacuo to the impure titled compound (2.81 g) as a yellowsolid. LCMS indicated the presence of two major diastereomers withretention times of 1.09, 1.18 minutes, respectively. Two minor isomerswere also observed. The product was stored under nitrogen at 4° C. untiluse in the following step without further purification. LCMS m/z 1068(M+H).

Intermediate 5(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

To a solution of above obtainedN-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ²-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(1.0 g, 0.936 mmol) in ethanol (5 mL) was added methylamine (33 wt % inethanol) (20 ml, 161 mmol) at room temperature. The mixture was stirredat room temperature for 30 minutes, after which time LCMS indicatedconsumption of starting materials. The reaction mixture was concentratedin vacuo to a tan residue. The material was taken into DMSO and purifiedusing chromatography (reverse phase silica gel, RediSep Gold C18, 30 g).Gradient elution was run at 100% water (with 0.1% NH4OH modifier) for 4CV followed by 0-15% acetonitrile in water (with 0.1% NH4OH modifier)for 3 CV followed by 15-25% acetonitrile in water (with 0.1% NH4OHmodifier) for 6 CV followed by 25-90% acetonitrile in water (with 0.1%NH4OH modifier) for 3 CV followed by an isocratic hold at 90%acetonitrile in water (with 0.1% NH4OH modifier) for 4 CV.

Fractions from the purification were combined and two major isomericproducts were isolated:

-   -   Isomer 2 of the titled compound as a bisammonium salt, with the        exact stereochemistry at two phosphorus centers undetermined (28        mg, purity by LCMS =54%; 0.018 mmol, 1.921% yield) as a        colorless resiude, LCMS m/z 807.1 (M+H), t_(RET)=0.80 min.    -   Isomer 3 of the titled compound as a bisammonium salt, with the        exact stereochemistry at two phosphorus centers undetermined (46        mg, purity by LCMS =70%, 0.038 mmol, 4.09% yield) as a yellow        residue, LCMS m/z 807.2 (M+H), t_(RET)=0.91 min.

Examples 8a and 8b(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

Isomer 2 of Intermediate 5,(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(25 mg, 0.031 mmol), obtained from the preceding step, was suspended inpyridine (0.5 mL) and triethylamine (0.5 mL). The mixture was stirredand heated to 50° C., then triethylamine trihydrofluoride (0.5 mL, 3.07mmol) was added and the mixture was stirred at 50° C. for 2 hours, afterwhich time, LCMS indicated complete consumption of starting material andconversion to desired product. The reaction mixture evaporated in vacuo,and the flask was stored under nitrogen at 4° C. overnight.

After warming to room temperature, the residue was taken into water (˜5mL) and a precipitate formed. The solids were separated from themixture, and the filtrate was adjusted to pH=10 using ammoniumhydroxide. The solution was purified using reverse phase HPLC (0-5%acetonitrile:water (with 0.1% NH4OH modifier), C18 50×30 mm Geminicolumn, 40 mL/min, 7 minute gradient, UV detection=214 nm). Thecollected solids were taken into water (˜2 mL) and methanol (˜0.5 mL),and ammonium hyroxide was added to pH=10. The solution was purifiedusing reverse phase HPLC (0-5% acetonitrile:water (with 0.1% NH4OHmodifier), C18 50×30 mm Gemini column, 40 mL/min, 7 minute gradient, UVdetection=214 nm).

The desired fractions from both purifications were combined andevacuated in vacuo to afford a colorless residue. The residue was takeninto water (2 mL) and lyophilized overnight to afford the titledcompound (Example 8a, 7 mg) as a bisammonium salt as a singlediastereomer, with the exact stereochemistry at the two phosphoruscenters undetermined. The product was a white solid. LCMS m/z 693.3(M+H). t_(RET)=0.29 min.

¹H NMR (400 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 8.59-8.62 (m, 1H),8.39-8.41 (m, 1H), 8.17 (s, 1H), 8.13 (s, 1H), 6.19-6.27 (m, 1H),6.05-6.10 (m, 1H), 5.70-5.87 (m, 1H), 5.24-5.36 (m, 1H), 5.14-5.23 (m,1H), 4.31-4.36 (m, 1H), 4.23-4.29 (m, 1H), 4.15-4.23 (m, 1H), 4.11-4.15(m, 1H), 3.98-4.08 (m, 1H), 3.82-3.91 (m, 1H), 3.66-3.72 (m, 1H).

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 53.66, 55.91.

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −206.48.

Isomer 3 of Intermediate 5,(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(175 mg, 0.217 mmol), obtained from a different batch (purity byLCMS=50%), was suspended in pyridine (1 mL) and triethylamine (1 mL).The mixture was stirred and heated to 50° C., then triethylaminetrihydrofluoride (1 mL, 6.14 mmol) was added and the mixture was stirredat 50° C. for 3 hours, after which time, LCMS indicated completeconsumption of starting material and conversion to desired product. Themixture was allowed to cool to room temperature, then acetone (˜10 mL)was added and a fine precipitate formed. The precipitate was collectedvia vacuum filtration to afford a gray residue, which was discarded. Thefiltrate was evaporated in vacuo, and the flask was stored undernitrogen at 4° C. overnight.

After warming to room temperature, the residue was taken into methanol(˜6 mL) and purified using reverse phase HPLC (0-15% acetonitrile:water(with 0.1% NH4OH modifier), C18 50×30 mm Gemini column, 47 mL/min, 8minute gradient, UV detection=214 nm). The desired fractions werecombined and evacuated in vacuo to afford a white solid, stillcontaminated by some impurities.

The solid was further purified using preparative HILIC column (LunaHILIC, 5 u 21×250 mm, 20 mL/min, UV detection=254 nm) with an isocraticgradient of 20% aqueous ammonium formate and 80% acetonitrile. Thedesired fractions were combined and evaporated in vacuo 90%, then takeninto water and acetonitrile and 5 drops of ammonium hydroxide was addedto pH=10. The material was frozen and lyophilized overnight. Thisprocess was repeated twice more to afford the titled compound (Example8b, 12 mg) as a bisammonium salt as a single diastereomer, with theexact stereochemistry at the two phosphorus centers undetermined. Theproduct was a white solid. LCMS m/z 693.3 (M+H), t_(RET)=0.37 min.

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 8.50-9.29 (m, 1H),8.43 (br s, 1H), 8.16 (s, 1H), 7.73-8.11 (m, 1H), 6.26 (br d, J=14.4 Hz,1H), 6.15 (br d, J=7.2 Hz, 1H), 5.72 (s, 1H), 5.29-5.41 (m, 1H),5.17-5.29 (m, 1H), 4.27-4.46 (m, 2H), 4.21 (br s, 1H), 4.01-4.15 (m,1H), 3.86-3.91 (m, 1H), 3.81-3.86 (m, 1H), 3.77 (br d, J=10.6 Hz, 1H).

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 54.27, 49.69.

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −204.90 (br.)

Examples 9a and 9b Compounds 2a and 2b(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

Intermediate 2(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

Intermediate 2 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 2 inother Examples, may be used.

To a room temperature solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite (4.01 g, 4.6 mmol) in acetonitrile (35 mL)and water (0.165 mL, 9.1 mmol) was added pyridine 2,2,2-trifluoroacetate(1.06 g, 5.5 mmol). The reaction was stirred for 10 minutes then neattert-butylamine (24.2 mL, 228 mmol) was added. The reaction was stirredat room temperature for 30 minutes then concentrated under reducedpressure. The resulting white foam was azeotroped from acetonitrile (2×)then the dry residue was taken into a mixture of dichloromethane (100mL) and water (0.82 mL, 45.7 mmol) and treated with neat2,2-dichloroacetic acid (3.01 mL, 36.5 mmol). After 30 minutes, thereaction was quenched with pyridine (5.91 mL, 73.1 mmol) thenconcentrated under reduced pressure to an oily suspension. The materialwas azeotroped from acetonitrile (3×) then taken into anhydrousacetonitrile (60 mL) and concentrated to a volume of about 20 mL toafford the impure titled compound as a light orange suspension. LCMS m/z437.9 (M+H). This mixture was used in the next step without furtherpurification.

Intermediate 3(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogenphosphonate

Intermediate 3 was made generally according to the procedure below.Slight modifications, for example those depicted for Intermediate 3 inother Examples, may be used.

(2R,3R,4R,5R)-2-(6-Benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (5.9 g, 5.9 mmol) wasazeotroped from acetonitrile (2×) then taken into 40 mL of anhydrousacetonitrile, concentrated by about half, then stored under nitrogenover 3A molecular sieves. After 1 hour, this solution was added to thepreviously prepared crude mixture (Intermediate 2) of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (2.0 g, 4.6 mmol) under nitrogen. The reaction wasstirred at room temperature for 1 hour then was treated with3-(dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione (1.03 g,5.0 mmol), stirred for 30 minutes and concentrated under reducedpressure. The residue was taken into a mixture of dichloromethane (60mL) and water (0.823 mL, 45.7 mmol) then treated with 2,2-dichloroaceticacid (4.5 mL, 54.8 mmol). This was stirred at room temperature for 15minutes before quenching with pyridine (25 mL, 309 mmol) andconcentration under reduced pressure. The oily concentrate wasazeotroped from pyridine then taken into anhydrous pyridine (60 mL) andconcentrated under reduced pressure to about 20 mL to afford the impuretitled compound as a dark orange oil. LCMS m/z 1054.2 (M+H). Thismixture was used in the next step without further purification.

Intermediate 6N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide

To a crude solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogenphosphonate (Intermediate 3, 4.8 g, 4.6 mmol) in pyridine (20 mL) undernitrogen was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide(3.0 g, 16.0 mmol). The reaction stirred at room temperature undernitrogen for 30 minutes then was quenched with the addition of water(2.9 mL, 160 mmol) followed by iodine (1.5 g, 5.9 mmol). After 10minutes, the mixture was poured into a solution of sodium bisulfite(0.95 g, 9.1 mmol) in water (300 mL). After 5 minutes, the reactionmixture was treated portion-wise with solid sodium bicarbonate (19.2 g,229 mmol). The resulting tan suspension was extracted with EtOAc (3×200mL) then the extracts were washed with saturated aqueous sodiumbicarbonate, dried over Na₂SO₄ and concentrated to an oil. One of thetwo following purification methods was then used to purify the productfor different batches.

Method A: The oil was azeotroped from toluene to remove excess pyridinethen purified by chromatography on silica (Biotage-100 g) eluting withsuccessive gradients of 0-10% MeOH in DCM (10 min), 10% MeOH in DCM (10minutes), 10-20% MeOH in DCM (10 min) and finally 20-40% MeOH in DMC (10min). The fractions of interest identified by LCMS were combined andconcentrated to afford the impure titled compound (1.03 g, 0.979 mmol)as a light orange solid. Two isomers were observed by LCMS [m/z 1052.3(M+H)] in an approximate ratio of 1:1 with retention times of 1.00, 1.09minutes, respectively. The product was stored under nitrogen at 4° C.and used in the next step without further purification.

Method B: The oil was azeotroped from toluene to remove excess pyridinethen purified by chromatography on silica (Teledyne ISCO Gold—120 g)eluting with successive gradients of 100% DCM (5 min), 0-10% MeOH in DCM(5 min), 10% MeOH in DCM (10 minutes), and 10-40% MeOH in DMC (20 min).The fractions of interest were combined and concentrated to afford anapproximate 1:1 mixture of diastereomers as a dark yellow solid. Themixture of diastereomers was separated by reverse phase HPLC (GeminiC-18: 30×50 mm column; 45-60% CH₃CN w/0.1% TFA/water w/0.1% TFA), 12 minrun collecting at 214 nm. The fractions of interest were combined,treated with saturated aqueous sodium bicarbonate then concentrated toremove acetonitrile. The aqueous concentrates were then extracted withEtOAc. The dried extracts (over Na₂SO₄) were concentrated to afford theindividual diastereomers.

-   -   Diastereomer 1 of the titled compound (42 mg) as a white solid,        with the exact stereochemistry at the chiral phosphorus center        undetermined. LCMS m/z 1052.7 (M+H), t_(RET)=1.00 min.    -   Diastereomer 2 of the titled compound (43 mg) as a white solid,        with the exact stereochemistry at the chiral phosphorus center        undetermined. LCMS m/z 1052.7 (M+H). t_(RET)=1.09 min.

Examples 9a and 9b(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

A solution of intermediate 6 (purified by method A),N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(1.03 g, 0.979 mmol) in 33% wt methylamine in EtOH (40 mL, 321 mmol) wasstirred at room temperature for 4 hours whereupon LCMS indicatedcomplete conversion of the starting material and the presence of thedesired O-TBS protected intermediate. Only one, slightly broad peak forthe intermediate was noted, indicating at least one diastereomer waspresent. The reaction was concentrated to a dark orange residue whichwas purified by reverse phase HPLC (Gemini C-18: 30×50 mm column; 10-60%acetonitrile w/0.1% TFA/water w/0.1% TFA), 12 minute gradient withdetection at 254 nm. The fractions of interest were combined andconcentrated under reduced pressure. At this point, LCMS indicated theloss of the silyl protecting group. The aqueous phase was furtherconcentrated to approximately 3-5 mL then methanol (25 mL) was added.The resulting suspension was filtered and the solids were rinsed withMeOH and then diethyl ether then suction dried to afford 75 mg of theimpure desired product-diTFA salt as a white solid. LCMS m/z 677.2(M+H).

The product was further purified by prep-chromatography (Luna HILIC 3 u:4.6×150 mm; isocratic 20% 30 mM aqueous HCO₂NH₄, 80% CH₃CN). Thefractions of interest were combined and concentrated to a residue thatwas lyophilized out of water (5 mL) and 3 drops ammonium hydroxide). Toremove residual ammonium formate, the lyophilization process wasrepeated 4 more times to afford the titled compound (Example 9a, 50 mg)as a bisammonium salt as a single diastereomer, with the exactstereochemistry at the chiral phosphorus center being undetermined. Thetitled product was a white solid. LCMS m/z 677.6 (M+H). t_(RET)=0.11 min

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.48 (s, 1H), 8.39(s, 1H), 8.17 (s, 1H), 8.12 (s, 1H), 6.23 (dd, J=15.1, 3.0 Hz, 1H), 6.09(d, J=8.3 Hz, 1H), 5.69 (br dt, J=52.1, 3.4 Hz, 1H), 5.16 (ddd, J=8.1,6.6, 4.2 Hz, 1H), 5.02-5.10 (m, 1H), 4.35 (d, J=4.2 Hz, 1H), 4.24 (br s,1H), 4.11-4.18 (m, 1H), 4.09 (br s, 1H), 3.97 (br d, J=10.6 Hz, 1H),3.89-3.95 (m, 1H), 3.72 (br d, J=12.5 Hz, 1H).

¹³C NMR (150 MHz DMSO-d₆, with one drop of D₂O): δ ppm 156.0, 155.8,153.0, 152.8, 150.3, 148.9, 119.1, 118.4, 92.4, 85.4, 84.0, 83.3, 81.0,77.9, 72.3, 71.4, 65.9, 62.6.

³¹P NMR (162 MHz, DMSO-d₆, with one drop of D₂O) δ ppm 55.67 and −2.51.

¹⁹F NMR (376 MHz, DMSO-d₆, with one drop of D₂O) δ ppm −205.16.

It is noted that Example 9a may also be prepared using the proceduredepicted below for Example 9b.

A solution of Diastereomer 2 of Intermediate 6 (purified by method B),N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(43 mg, 0.041 mmol), in 33% wt methylamine in EtOH (8.0 mL, 64.3 mmol)was stirred at room temperature for 30 minutes and then concentrated.The resulting dark orange residue was taken into anhydrous pyridine(0.50 mL) and triethylamine (0.50 mL), heated to 50° C. then treatedwith triethylamine trihydrofluoride (0.50 mL, 3.07 mmol). After 1 hour,the reaction was complete. The mixture was cooled and concentrated to adark oil that was taken into water (7.5 mL) and ammonium hydroxide (10drops). The resulting suspension (pH-3) was filtered and the solids weredissolved into a mixture of water (2 mL) and ammonium hydroxide (1 mL)then purified by reverse phase HPLC (Gemini C-18: 30×50 mm column; 0-10%acetonitrile/water w/0.1% NH4OH; 214 nm). The fractions of interest werecombined and concentrated under reduced pressure to a wet residue thatwas taken into water (5 ml) and 5 drops ammonium hydroxide thenlyophillized to afford the titled compound (Example 9b, 7.0 mg) as abisammonium salt as a single diastereomer, with the exactstereochemistry at the chiral phosphorus center being undetermined. Theproduct was a white solid. LCMS m/z 677.2 (M+H), t_(RET)=0.32 min.

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.65-9.35 (m, 1H),8.44 (br s, 1H), 7.77-8.29 (m, 2H), 6.25 (br d, J=14.4 Hz, 1H),6.11-6.19 (m, 1H), 5.53-5.73 (m, 1H), 5.18-5.44 (m, 1H), 4.96-5.08 (m,1H), 4.40-4.54 (m, 1H), 4.33 (br s, 2H), 4.23-4.30 (m, 1H), 4.16 (br s,1H), 3.98-4.06 (m, 1H), 3.81 (br s, 1H).

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 49.48 and −2.94.

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −206.44 (br).

Examples 10a and 10b Compounds 28a and 28b(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione

Intermediate 7(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate

(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (4.0 g, 4.12 mmol) wasazeotroped with acetonitrile (20 mL) three times. After the lastconcentration, ˜15 mL of acetonitrile was kept in the reaction flask and3 Å molecular sieves (˜20 beads) were added to the clear solution. Thesolution was left standing over the molecular sieves under nitrogen for˜1 hour.

To a separate round bottom flask of the previously prepared crudemixture (Intermediate 2) of(2R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (1.5 g, 3.43 mmol) in acetonitrile (10 mL) wasadded via syringe the above pre-dried solution of(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite in acetonitrile (˜15 mL).After 30 minutes of stirring at room temperature,(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(DDTT) (780 mg, 3.80 mmol) was added. The mixture was stirred at roomtemperature for 30 minutes, followed by evaporation of the acetonitrilein vacuo. Dichloromethane (DCM) (50 mL) and Water (650 μL) were thenadded to the residue, followed by the addition of 2,2-dichloroaceticacid (3.5 mL, 42.4 mmol). This was stirred at room temperature for 30minutes before being quenched with pyridine (20 mL). The mixture wasconcentrated in vacuo to afford the impure titled compound as an orangeoil. LCMS m/z 1036.2 (M+H). The crude product was used in the next stepwithout further purification.

Intermediate 8N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide

To a crude solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate (Intermediate7, 3.55 g, 3.43 mmol) in pyridine (60 mL) was added2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (DMOCP) (2.2 g,11.92 mmol), and the mixture was stirred under nitrogen at roomtemperature for 30 minutes. The reaction was quenched with water (2.2mL, 10 equiv to DMOCP), followed by addition of iodine (1.2 g, 4.73mmol). The mixture was stirred for 10 minutes, then poured into asolution of water (400 mL) and sodium bisulfite (NaHSO₃) (1.0 g, 9.61mmol). After 5 minutes of stirring, sodium bicarbonate (NaHCO₃) (14.4 g,171 mmol) was slowly added portionwise as solid (caution: gasevolution). The product was extracted with 1:1 diethyl ether:EtOAc (300mL×2) and the combined extracts were dried over sodium sulfate,filtered, and concentrated in vacuo. Excess pyridine was removed byconcentration with toluene (100 mL×2). The crude material was purifiedby chromatography on silica (100 gram column) using a gradient of 0-20%MeOH/DCM, then holding at 20% MeOH/DCM until all the desired product hadeluted off the column. Desired fractions were combined and concentratedto afford two isomeric products:

-   -   Isomer 1 of the titled compound, more polar, as an impure        mixture (1.39 g, purity by LCMS ˜33% along with ˜28% of Isomer        2). LCMS m/z 1034.1 (M+H), t_(RET)=0.98 min.    -   Isomer 2 of the titled compound, less polar, as an impure        mixture (230 mg, purity by LCMS ˜33%). LCMS m/z 1034.2 (M+H),        t_(RET)=1.09 min.

Intermediates 9a and 9b(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]l-9-fluoro-12-hydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione

Isomer 1 of Intermediate 8,N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(1.39 g, 1.34 mmol), was stirred in methanamine (33% wt in EtOH) (10.0mL, 80 mmol) under nitrogen at room temperature for 3 hours. Thereaction mixture was concentrated and the residue was purified viareverse phase HPLC, using a gradient of 10-50% ACN/H₂O (0.1% TFA)), toafford Isomer 1 of the titled compound (Intermediate 9a, impure, 280 mg)as a tan solid. LCMS m/z 807.1 (M+H), t_(RET)=0.80 min.

Following the same procedure for the preparation of Intermediate 9a,Isomer 2 of Intermediate 8,N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(230 mg, 0.22 mmol), was stirred in methanamine (33% wt in EtOH) (2.0mL, 16 mmol) under nitrogen at room temperature for 3 hours. Thereaction mixture was concentrated and the residue was purified viareverse phase HPLC, using a gradient of 10-50% ACN/H₂O (0.1% TFA)), toafford Isomer 2 of the titled compound (Intermediate 9b, impure, 60 mg)as a tan solid.

LCMS m/z 807.1 (M+H), t_(RET)=0.85 min.

Examples 10a and 10b(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione

To a suspension of Isomer 1 of(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-12-hydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(Intermediate 9a, 280 mg, 0.35 mmol) in pyridine (2 mL) andtriethylamine (2 mL) at 50° C. was added triethylamine trihydrofluoride(1.5 mL, 9.21 mmol). The mixture was stirred at 50° C. for 4 hours. LCMSindicated some starting material still unconsumed, and the reaction wasleft to stir at room temperature for 16 hours. After that, the reactionmixture was diluted with acetone (˜25 mL) and precipitation was formed.After 15 minutes of stirring, the reaction was filtered. The solid wasrinsed with acetone (˜10 mL) and dried. The filtrate was concentrated invacuo and was then added toluene to remove any remaining pyridine. Thecrude filtrate and filtered solid were individually purified via reversephase HPLC, using a gradient of 0-20% ACN:H₂O (0.1% NH₄OH) and combined.¹⁹F NMR spectra displayed residual TFA present in the samples. A secondreverse phase HPLC purification, using a gradient of 0-10% ACN/H₂O (0.1%NH₄OH), afforded the titled compound (Example 10a, 4 mg) as abisammonium salt as a single diastereomer, with the exactstereochemistry at the phosphorus center undetermined. The product was awhite solid. LCMS m/z 693.0 (M+H). t_(RET)=0.11 min.

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.30 (s, 1H), 8.19(s, 1H), 7.97 (s, 1H), 6.26 (dd, J=15.9, 2.3 Hz, 1H), 5.86 (d, J=8.3 Hz,1H), 5.59-5.76 (m, 1H), 5.30 (br s, 1H), 5.06 (brd, J=15.5 Hz, 1H), 4.35(d, J=3.8 Hz, 1H), 4.25 (br s, 1H), 4.02-4.07 (m, 1H), 4.01-4.13 (m,2H), 3.88-3.99 (m, 2H).

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −203.83.

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 55.73, −2.66.

Following the same procedure for the preparation of Example 10a, thereaction of Isomer 2 of(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-12-hydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(Intermediate 9b, 60 mg, 0.35 mmol) afforded the titled compound(Example 10b, 7 mg) as a bisammonium salt as a single diastereomer, withthe exact stereochemistry at the phosphorus center undetermined. Theproduct was a white solid. LCMS m/z 693.0 (M+H), t_(RET)=0.37 min.

¹H NMR (400 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.50 (br. s., 1H), H) 8.22 (br. s., 1 H), 7.94 (br. s., 1H), 6.35 (d, J=14 Hz, 1H),5.87 (d, J=7.86 Hz, 1 H), 5.54-5.67 (m, 1 H), 4.98 (br. d., J=15.5 Hz, 1H), 4.39 (br. s., 1 H), 4.33 (d, J=6.84 Hz, 1 H), 4.24 (br. s., 1 H),4.13 (br. s., 1 H), 4.00-4.10 (m, 2 H), 3.89-3.98 (m, 2 H).

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −205.00.

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 49.15, −2.90.

Examples 11a and 11b Compounds 27a and 27b(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione, bisammonium salt

Intermediate 10N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}

To a crude solution of(2R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (Intermediate 7, 2.58 g, 2.49 mmol) in pyridine (50mL) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide(DMOCP) (1.70 g, 9.21 mmol), and the mixture was stirred under nitrogenat room temperature for 30 minutes. The reaction was then quenched withwater (1.6 mL, 89 mmol), followed by addition of3H-benzo[c][1,2]dithiol-3-one (660 mg, 3.92 mmol). The mixture wasstirred for 10 minutes, then poured into a beaker containing water (350mL) and sodium bicarbonate (NaHCO₃) (10 g, 119 mmol). The yellow slurrywas stirred for 10 minutes, then transferred to a separatory funnel. Theproduct was extracted with 1:1 diethyl ether:EtOAc (300 mL×2). Thecombined extracts were dried over sodium sulfate, filtered, andconcentrated in vacuo. The crude material was purified by chromatographyon silica (100 gram column) eluting with 0-10% MeOH/DCM, then holding at10% MeOH/DCM. The desired fractions were combined and concentrated toafford the impure titled compound (1.1 g) as a tan solid. Two majorisomers appeared to make up ˜77% of the mixture by LCMS. LCMS m/z 1050.1(M+H), t_(RET)=1.09 and 1.20 min, respectively.

Intermediate 11(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]l-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione

ImpureN-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-12-sulfanyl-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(Intermediate 10, 0.77 g, 0.73 mmol) was stirred in methanamine (33% wtin EtOH) (10.0 mL, 80 mmol) under nitrogen at room temperature for 2hours. Crude LCMS indicated 4 isomers ((M+H)⁺=823) with t_(RET)=0.74,0.83, 0.90, and 0.94 min, in a rough ratio of 20:3:13:19 (but the ratiocould be significantly affected by possible overlaps with impuritypeaks). The volatiles were removed in vacuo. The crude material waspurified via reverse phase HPLC, using a gradient of 10-60% ACN:H₂O(0.1% TFA). Two major isomers were separated.

-   -   Isomer 1 of the titled compound (120 mg, 64% purity by LCMS        along with 8% TBS-deprotected product) as a tan solid, with the        exact stereochemistry at two phosphorus centers undetermined.        LCMS m/z 823.1 (M+H). t_(RET)=0.74 min.    -   Isomer 2 of the titled compound (130 mg, 50% purity by LCMS        along with 18% TBS-deprotected product) as a tan solid, with the        exact stereochemistry at two phosphorus centers undetermined.        LCMS m/z 823.1 (M+H). t_(RET)=0.96-1.00 min as a broad peak with        peak tailing.

Examples 11a and 11b(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione,bisammonium salt

To a suspension of Isomer 1 of Intermediate 11,(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(120 mg, 0.146 mmol), in pyridine (2 mL) and triethylamine (2 mL) at 50°C. was added triethylamine trihydrofluoride (700 μL, 4.30 mmol) and themixture was stirred and heated at 50° C. for 4 hours. LCMS indicatedthat some starting material was still unconsumed. The reaction mixturewas left to stir for 16 hours at room temperature. Then acetone (˜25 mL)was added and solid precipitated out. This was left to stir at roomtemperature for ˜30 minutes, filtered and rinsed with acetone. Thefiltered solid did not contain any desired product by LCMS and wasdiscarded. The filtrate, containing the desired product, wasconcentrated in vacuo and toluene was added to further remove anyremaining pyridine. The residue was purified via reverse phase HPLC,using a gradient of 0-10% ACN:H₂O (0.1% NH₄OH), to afford the productthat was not very pure and appeared to be contaminated bytrifluoroacetate. So the solid was taken up in ˜2 mL of water and addedseveral drops of 30% aqueous NH₄OH. It was further purified via reversephase HPLC, using a gradient of 0-10% ACN:H₂O (0.1% NH₄OH), to affordthe titled compound (Example 11a, 13 mg) as a bisammonium salt as asingle diastereomer, with the exact stereochemistry at two phosphoruscenters undetermined. The product was a white solid. LCMS m/z 708.9(M+H). t_(RET)=0.17 min.

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.31 (s, 1H), 8.21(s, 1H), 8.11 (br s, 1H), 6.25 (dd, J=15.1, 2.6 Hz, 1H), 5.84 (d, J=8.3Hz, 1H), 5.68 (d, J=51.7 Hz, 1H), 5.27-5.37 (m, 1H), 5.16-5.25 (m, 1H),4.32 (d, J=4.2 Hz, 1H), 4.26 (br s, 1H), 4.01-4.17 (m, 3H), 3.90-3.96(m, 1H), 3.81 (br d, J=11.7 Hz, 1H).

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −205.30 (br).

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 55.77, 54.01.

Following the same procedure for the preparation of Example 11a, exceptthat the first purification used a gradient of 0-20% ACN:H₂O (0.1%NH₄OH), the reaction of Isomer 2 of Intermediate 11,(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione(130 mg, 0.158 mmol), afforded the titled compound (Example 1 b, 16 mg)as a bisammonium salt as a single diastereomer, with the exactstereochemistry at two phosphorus centers undetermined. The product wasa white solid. LCMS m/z 708.9 (M+H). t_(RET)=0.42 min.

¹H NMR (600 MHz, DMSO-d₆ with one drop of D₂O): δ ppm 8.22 (br s, 1H),8.18 (s, 1H), 8.05 (br s, 1H), 6.27 (dd, J=15.3, 2.1 Hz, 1H), 5.82 (brd, J=8.3 Hz, 1H), 5.60 (d, J=49.9 Hz, 1H), 5.27-5.46 (m, 1H), 5.12-5.27(m, 1H), 4.42-4.59 (m, 1H), 4.30 (br s, 1H), 4.14 (br d, J=2.3 Hz, 1H),4.11 (br d, J=5.7 Hz, 2H), 4.06 (br d, J=9.1 Hz, 1H), 3.82 (br d, J=11.0Hz, 1H).

¹⁹F NMR (376 MHz, DMSO-d₆ with one drop of D₂O) δ ppm −205.05.

³¹P NMR (162 MHz, DMSO-d₆ with one drop of D₂O) δ ppm 53.85, 47.48.

Abbreviations

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

-   DCM Dichloromethane-   DMF N, N-Dimethylformamide-   DMSO Dimethylsulphoxide-   DMTr Dimethoxytrityl-   THF Tetrahydrofuran-   EtOAc Ethyl acetate-   MeOH Methanol-   EtOH Ethanol-   MeCN Acetonitrile-   HCl Hydrochloric acid-   HPLC High performance liquid chromatography-   MDAP Mass Directed Autopreparative HPLC-   SPE Solid phase extraction-   MeOH Methanol-   TBDMS tert-Butyldimethylsilyl-   TBME tert-Butyl methy ether-   TFA Trifluoroacetic acid-   DIPEA N, N-Diisopropylethylamine

Nomenclature

The compounds were named from the structure using either thenomenclature tool in Chem Draw (CambridgeSoft) or Marvin Sketch(ChemAxon).

Example 12 Injectable Composition

An injectable form for administering the present invention is producedby stirring 1.7% by weight of Compound #2 in a 0.9% saline solution.

Assay

The compounds are tested in a STING binding assay similar to thatdescribed by Li et al. (Nature Chemical Biology, 10, 1043-1048, (2014)).

Biological Activity

Compounds of the invention were tested in a STING binding assay similarto the one described in Li et al. (Nature Chemical Biology, 10,1043-1048, (2014)). Compounds of the invention were tested in aFluorescence Resonance Energy Transfer (FRET) binding assay. Li et al.used a Scintillation Proximity Assay (SPA) binding assay.

STING activity for compounds of the invention is listed in Table 1below.

TABLE 1 Example Compound STING FRET pIC50  8a  1a 5.2  8b  1b 6.2  9a 2a 4.9  9b  2b 6.0 11a 27a 7.3 11b 27b 8.4 10a 28a 6.9 10b 28b 8.3

While the preferred embodiments of the invention are illustrated by theabove, it is to be understood that the invention is not limited to theprecise instructions herein disclosed and that the right to allmodifications coming within the scope of the following claims isreserved.

The invention claimed is:
 1. A compound of Formula (VI):

wherein: X⁵¹ is S; X⁵² is S; R⁵¹ is OH and R⁵² is NH₂ or R⁵¹ is NH₂ andR⁵² is H; R⁵³ is OH and R⁵⁴ is NH₂ or R⁵³ is NH₂ and R⁵⁴ is H; R⁵⁵ isselected from: F and OH; R⁵⁶ is F; R⁵⁸ and R⁵⁹ are independentlyselected from: H, CH₂OC(O)R⁵⁷, CH₂OCO₂R⁵⁷, CH₂CH₂SC(O)R⁵⁷, andCH₂CH₂SSCH₂R⁵⁷; where R⁵⁷ is selected from: aryl, heteroaryl,heterocycloalkyl, cycloalkyl, C₁₋₂₀alkyl and C₁₋₂₀alkyl substituted withone to 5 substituents independently selected from: aryl, cycloalkyl,hydroxy and F; provided at least one of R⁵⁸ and R⁵⁹ is not H, andprovided that none of R⁵⁸ and R⁵⁹ is —CH₂OC(O)C(CH₃)₃, or apharmaceutically acceptable salt thereof.
 2. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: R⁵⁵ is OH.
 3. Acompound of claim 1 selected from:

or a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and one or more of pharmaceutically acceptableexcipients.
 5. A method of treating a disease state selected from:inflammation, allergic diseases, autoimmune diseases, humanimmunodeficiency virus (HIV), infectious diseases, cancer, andpre-cancerous syndromes, in a mammal in need thereof, which comprisesadministering to such mammal a therapeutically effective amount of acompound 1, or a pharmaceutically acceptable salt thereof.
 6. The methodof claim 5 wherein the mammal is a human.
 7. A composition comprising acompound of claim 1, or a pharmaceutically acceptable salt thereof, andan antigen or antigen composition.
 8. A vaccine composition comprisingan antigen or antigen composition and a compound of claim 1, or apharmaceutically acceptable salt thereof.
 9. An immugenic compositioncomprising an antigen or antigen composition and a compound of claim 1,or a pharmaceutically acceptable salt thereof.
 10. A compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof, and one or more immunostimulatory agents.
 11. A method oftreating a disease selected from: HBV, HCV, influenza, skin warts,multiple sclerosis, and allergic inflammation, in a human in needthereof, which comprises administering to such mammal a therapeuticallyeffective amount of a compound, or a pharmaceutically acceptable saltthereof, as described in claim 1.